Tissue culture and advanced crop improvement using biotechnology

1. Assignment
Subject : Advanced Plant Breeding System (GPB903)
Presented by: Mr. Indranil Bhattacharjee
Student I.D. No.: 17PHGPB102
Presented to : Prof. (Dr.) S. Marker
Sam Higginbottom University of Agriculture, Technology &
Sciences
Allahabad-211007
Tissue Culture and Modern
Methods of Crop Improvement

2. Contents
 Introduction
 Historical background
 Requirement of Tissue Culture
 Major steps involved in Tissue Culture
 Why Tissue culture ?
 Major Techniques and its Achievements
 Applications
 Conclusion

3. What is Tissue Culture?
 Tissue culture is the term used for “The
process of growing cells artificially in the
laboratory under controlled conditions”
 Tissue culture is possible in both plant and
animal cells and the products identical
plants/animals, in which all product cells have
the same genotype (unless affected by
mutation during culture)

4. What’s the Background?
 Tissue culture had its
origins at the beginning
of the 20th century with
the work of Gottleib
Haberlandt (plants) and
Alexis Carrel (animals)
Haberlandt
Carrel

5. Historical
 The first commercial use of
plant clonal propagation on
artificial media was in the
germination and growth of
orchid plants, in the 1920’s
 In the 1950’s and 60’s there
was a great deal of
research, but it was only
after the development of a
reliable artificial medium
(Murashige & Skoog, 1962)
that plant tissue culture
really ‘took off’ commercially
Young plants of orchid

6.  A more recent advancement is the use of
plant and animal tissue culture along with
genetic modification using bacterial vectors
and gene guns to create genetically
engineered organisms (GE)

7. Plant Tissue Culture: historical highlights
1902 : Haberlandt attempted to the culture mesophyll tissue and root hair cells. This was the first
attempt of in vitro culture.
1904 : Haning attempted to culture excised embryos from mature seeds.
1922 : Kotte was successful in obtaining growth from isolated root tips on inorganic media.
Robbins reported similar success from root tip and stem tip.
1920-40 : First PGR, IAA, discovered by experiments on oat seedlings (Fritz Went).
1934 : Used yeast extract (vit B) with inorganic salts to repeatedly culture root tips of tomato.
1935 : Importance of B vitamins and PGRs in culture of mesophyll cells.
1936 : Haning experiment was repeated with IAA: works !!!
1939 : Tobacco crown gall culture, callus obtained: called as Plant Cancer.
1950s : Skoog used adenine sulfate to obtain buds on tobacco segments: PGR #2 identified: kinetin
1958 : Stewart and Reinert obtained somatic embryos from carrot cells using PGRs.
1950-60s : Botanists turned to plant tissue culture to study plant development.

8.  1960 : Cocking isolated protoplasts from cultured cells.
 1962 : Murashige and Skoog developed MS media for tobacco.
 1966 :Guha and Maheshwari obtained first haploid plants (D.U., India)
 1970 :Discovery of Restriction Endonuclease (Daniell Nathan).
 1972-73: First recombinant molecule created by Stanley Cohen,
Stanford Univ.
 1974 : Discovery of Ti plasmid in Agrobacterium tumefaciens (by
Zaenen in Ghent Univ., Belgium)
 1970-80s:Ti plasmid analysis (Nester, Seattle; Van Montagu, Ghent)
 1983 : First Transgenic Plant (Monsanto, Ghent, Washington Univ).
 1985 : Leaf disk transformation method (Monsanto)

9. Plant Genetic Engineering
1. Plant Tissue Culture
2. Plant Molecular Biology
3. Plant Genetics
 What made Biotechnology Possible:
 Ability to recover regenerated plants from tissue and organ
culture [Tissue culture provided another level of genetic
variation: somaclonal variation].
 Ability to cut and ligate DNA: gene mapping and cloning
techniques.
 Ability to introduce foreign DNA that ends up in the nucleus and
ligates with the native DNA.

10. Plant cells are totipotent
 Totipotency: ability of a cell or tissue or organ to
grow and develop into a fully differentiated
organism.

11. Tissue Culture:
De-differentiation to Regeneration
seeds
Wheat inflorescence
+ auxinCallus- auxin/ + cytokinin
embryos

12. The Requirements?
 Appropriate tissue (Apical bud, meristemic
cells, anther, ovule, protoplast or cytoplasm
etc.).
 A suitable growth medium containing energy
sources and inorganic salts to supply cell
growth needs. This can be liquid or semisolid.
 Aseptic (sterile) conditions as microorganisms
grow much more quickly than plant and animal

13. Growth regulators
 In plants, both auxins & cytokinins
 In animals, this is not as well defined and the
growth substances are provided in serum from the
cell types of interest.
Frequent subculturing
To ensure adequate nutrition and to avoid the build
up of waste metabolites

14. Plant Tissue Culture- Major Steps
(1) Selection of the plant tissue
(Explant): any living tissue: apical
bud, leaf, root, zygotic embryo or
any other tissue of the healthy
vigorous ‘mother plant’.
(2) Sterilization: explant or tissue
must be sterilized to remove
microbial contaminants.

15. (3) Establishment of the explant in a
culture medium. The medium sustains
the plant cells and encourages cell
division. It can be solid or liquid.
Each plant species (and sometimes the
variety within a species) has particular
medium requirements that must be
established by trial and error.

16. (4) Multiplication- The explant gives
rise to a callus (a mass of loosely
arranged cells) which is
manipulated by varying sugar .
Concentrations and the auxin
(low): cytokinin (high) ratios to
form multiple shoots.
 The callus may be subdivided a
number of times.

17. (5) Root formation -
The shoots are
transferred to a
growth medium with
relatively higher
auxin: cytokinin
ratios

18. (6) Hardening
 The rooted shoots are
potted up (deflasked)
and ‘hardened off’ by
gradually decreasing
the humidity
 This is necessary as
many young tissue
culture plants have no
waxy cuticle to prevent
water loss

19. Callus
(Mass of Parenchymatous cells)
Organogenesis Somatic embryogenesis
Plant regeneration

20. 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

21. Non-zygotic embryogenesis or somatic embryogenesis
Explant Callus Embryogenic callus Somatic embryo Plant
2,4-D BA, zeatin PGR/ noPGR
Dicot : Globular, heart, torpedo and cotyledonary stages
Monocot: Globular, scutellar and coleoptilar stages.

23. Why do Plant Tissue Culture?
(1)Fast Process: A single explant can be
multiplied into several thousand plants in less
than a year - this allows fast commercial
propagation of new cultivars.
(2) Safety: Rare and Endangered plant spp can
be cloned safely as taking an explant from
mother plant does not usually destroy the
mother plant.
(3) Continous Supply: Once established, a plant
tissue culture line can give a continuous supply

24. (4) Disease free plants: In plants prone to virus
diseases, virus free explants (new meristem tissue is
usually virus free) can be cultivated to provide virus
free plants
(5) Preservation: Plant ‘tissue banks’ can be frozen,
then regenerated through tissue culture
(6) Easy Transportation: Plant cultures in approved
media are easier to export than are soil-grown plants,
as they are pathogen free and take up little space
(most current plant export is now done in this manner)

25. (7)Shortening Breeding Cycle:Tissue culture
allows fast selection for crop improvement -
explants are chosen from superior plants, then
cloned.
(8) True to type: Tissue culture clones are ‘true
to type’ as compared with seedlings, which
show greater variability

26. Techniques of tissue culture and their
achievements
1. Protoplast culture
2. Haploid culture
3. Micropagation

27. Protoplasts
Landmark:
1960: E. C. Cocking (Univ Nottingham) isolated
protoplasts by treating explants with concentrated
cellulase isolated from a fungus. [Commercial
cellulase and macrozyme were not available till
1968].
Protoplast fusion
1. Somatic hybrids
2. Cybrids
Tobacco protoplasts

28. Inter-specific fusions
Datura innoxia X D. stramonium = D. straubii (O. Schieder)
Tomato X Kartoffel = Tomoffel (G. Melchers)
2n x 2n
4n
2n
Synkaryon
Heterokaryon
Somatic hybrids
Fusion of haploid protoplasts (derived from anther cultures)
n + n= 2n
Cybrid Technology
Mixing two cytoplasms without hybrid formation

29. Hybrids obtained through protoplast fusion
Symmetric or near symmetric hybrids
Solanum tuberosum + Lycopersicon esculeum
Datura innoxia + Atropa belladona
Arabidopsis thaliana + Brassica campestris
Arabidopsis thaliana + Nicotiana chinensis
Asymmetric hybrids (having somatic complement of only one species)
Daucus carota + Oryza sativa
Daucus innoxia + Physalis minima
Nicotiana tabacum + Daucus carota

30. Haploid Culture
Haploid plant (n) = recessive mutations displayed
n+n= double haploid
Occur spontaneously in inter-specific cross or induced by irradiating
pollen prior to pollination. Extremely poor efficiency.
Landmark
1964 Guha and Maheshwari cultured Datura innoxia anthers and
found that large portion of culture contains haploid cells.
Later: Microspore cultures.

31. Varieties developed through
Anther culture
Wheat : Hua Pei 1, Lung Hua 1, Jinghua 1, Yunhua
1 & 2
Rice : Tanfeng 1, Tan Fong 1, Hua Yu 1 & 2
Xhonghua 8 & 9
Tobacco: Tan Yu 1, Tan Yu 2 & 3, F 211
Barley : Mingo, Gwylan
Production of homozygous DH lines

32. Micropropagation
 In the practice of plant tissue culture,
microorganisms are called “contaminants”
because of their harmful effects on plant growth in
vitro.
 Six potential sources of contamination in the plant
tissue culture lab are:
 Air, Water, Growth Media, People, Equipment and
Plant Material

33. 1. A single node will produce a shoot
within 4-6 weeks that has 4-6 nodes.
2. Each plantlet can be "subcultured" to
produce another 4-6 plants each.
3. Hundreds to thousands of plants could
be developed from one node
4. Since these are produced from axillary
buds, the plantlets will be clones of the
mother plant.

34. Advantages of Micro-propagation
(1) Economical in time and space
(2) Greater output : can produce millions of uniformly
flowering and yielding plants
(3) Disease free
(4) Elite plants with exceptional characteristics
(5) High Net Return
(6) Facilitates safer movements of germplasm across nations
In vitro germplasm assures the exchange of pest and
disease free material

35. (7) Great for vegetatively reproduced crops and crops,
which produce few seeds or highly heterozygous
seeds.
Achievements:
Mass multiplication of Banana, Production of artificial
seeds in Eucalyptus, regeneration in Conifer trees are
some of the notable examples of micropropagation.

36. Regeneration
 The process whereby a part of a plant can be
turned into a whole new plant.
Leaf sprouting new shoots

37.  Regeneration is possible because plant cells
can be made totipotent using hormones.
 Differentiated tissue: stems, leaves, roots,
etc.
 Undifferentiated (embryonic) cells are
totipotent: can become a whole new plant by
differentiating into a whole new plant.

38. Steps involved in Tissue Regeneration
(1) Sterilization: Tissue must be sterile-
completely free of any microoganisms; done
using aseptic technique
(2) Differentiation: Starting tissue is called
an ex-plant., differentiated cells (these cells
have developed to be part of specialized
tissue (root, leaf, stem, ovary, cotyledon,
etc.).

39. (3) Transfer of explants are plated on a sterile
petridish containing hormones and
nutrients that promote the explant cells
to develop into callus.
(4) Callus development: - a mass of
undifferentiated cells developed into
seedlings.

40. (5) Transfer of callus cells to petridishes:
Individual cells (or clumps of cells) of the
callus are transferred aseptically to a
different petri dish containing sterile medium
that encourages the undifferentiated callus
cells to become shoots and roots.
One mass of callus cells can be divided and
transferred to many plates for development into
shoots and roots.

42. Once shoots and roots have developed, they are
transferred to soil and grown to maturity

44. A Genetically Engineered Plant has a New Gene
• Squash gets coat proteins from cucumber
virus to prevent viral disease (to vaccinate
the squash)
• Corn gets a gene from bacteria that allows it
to survive in the presence of weed killer
(one that is less harmful to environment that
other herbicides).
• Corn gets a gene from different bacteria that
allows it to produce its own pesticide
(protects against insects).

45. Applications of Tissue Culture in Crop Improvement
(1) Creation of variation:
Tissue culture has been exploited to create genetic variability
from which:
 Crop plants can be improved
 Improve the state of health of the planted material
 Increase the number of desirable germplasms
available to the plant breeder
 Incorporate specific traits through gene transfer
(Molecular Techniques)

46. 2. Production of haploids (rice, wheat and barley)
3. Triploid production (fruits and poplar)
4. Embryo Rescue/ Wide hybridization (numerous examples)
5. Somatic hybridization (scientific examples, few commercial
products)
6. 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.)

47. (7) Production of disease free plants
(8) Clonal propagation
(9) Secondary metabolite production (eg.Taxol production from
cell cultures derived from the bark cuttings of pacific yew tree)
(10) Synthetic seed
(11) Pathogen eradication
(12) Germplasm conservation (cryopreservation)
(13) Cytoplasm transfer and developments of cybrids.

48. (14) Large scale multiplication
(15) Breaking dormancy
(16) Overcoming self sterility
(17) Early flowering

49. GM Foods

50. “Transgenics” or GMOs are
defined as those organisms with
a gene or genetic construct of
interest that has been introduced
by molecular or recombinant
DNA techniques

51. • The power of this technique lies in
its ability to move genes from one
organism to crop plants to impart
novel characteristics
• It is possible to transfer genetic
material from algae, bacteria,
viruses or animals to plants or to
move genes between sexually
incompatible species
Transgenics

53. Gene gunGene gun

54. Transformation by Gene gun

55. Application of GM technology
 Improving yield
 Nutritional improvement
 Increasing shelf life of fruits and vegetables by
delayed ripening
 Conferring resistance to insects, pests and
viruses
 Tolerance to abiotic stresses (drought, salt,
water-logging)
 Herbicide tolerance
 Edible vaccines

57. Control
Flaver Saver

60. GM crops : Global status (2005)
 Developing countries : 11
 Industrial countries : 10
 Countries joining the GM club in 2005
(Iran, Portugal, France & Czech Republic)
 No. of EU countries growing GM (2005) : 3 to 5
(Spain & Germany)
 No. of farmers growing GM crops globally :
8.5 million
 No. of Indian farmers growing GM crops :
1 million
 First triple gene product (maize) released in US
in 2005

61. Global area of Biotech crops in 2005
(Million hectares)
S. No. Country Area Crops
1. USA 49.8 (55%) Soybean, Maize, Cotton, Canola,
Squash, Papaya
2. Argentina 17.1 (19%) Soybean, Maize, Cotton
3. Brazil 9.4 (10%) Soybean
4. Canada 5.8 (6%) Canola, Maize, Soybean
5. China 3.3 (4%) Cotton
6. Paraguay 1.8 (2%) Soybean
7. India 1.3 (1%) Cotton
8. South
Africa
0.5 (1%) Maize, Cotton, Soybean
9. Uruguay 0.3 (< 1%) Maize, Soybean
10. Australia 0.3 (< 1%) Cotton

62. Global area of Biotech crops in 2005
(Million hectares)
S. No. Country Area Crops
11. Mexico 0.1 (< 1%) Soybean, Cotton
12. Romania 0.1 (< 1%) Soybean
13. Philippines 0.1 (< 1%) Maize
14. Spain 0.1 (< 1%) Maize
15. Colombia < 0.1 Cotton
16. Iran < 0.1 Rice
17. Honduras < 0.1 Maize
18. Portugal < 0.1 Maize
19. Germany < 0.1 Maize
20. France < 0.1 Maize
21. Czech Republic < 0.1 Maize

65. Dominant Biotech Crops, 2005
S.
No.
Crop MHa %Biotech
1 Herbicide tolerant soybean 54.4 60
2 Bt maize 11.3 13
3 Bt/herbicide tolerant maize 6.5 7
4 Bt cotton 4.9 5
5 Herbicide tolerant Canola 4.6 5
6 Bt/herbicide tolerant cotton 3.6 4
7 Herbicide tolerant maize 3.4 4
8 Herbicide tolerant cotton 1.3 2
Total 90.0 100%

67. Global value of Biotech crop market
Year Value (Million $US)
1996 115
1997 842
1998 1973
1999 2703
2000 2734
2001 3235
2002 3656
2003 4152
2004 4663
2005 5248
Total 29321

68. VEGETABLES
Tomato, Potato, Eggplant
Lettuce, Celery, Cauliflower
Cabbage, Sugarbeet, Carrot,
Cucumbers, Sweetpotato,
Cassava
FRUITS
Apple, Strawberry,
Walnut, Muskmelon,
Papaya, Grape
Transgenic Crops
for Food
EDIBLE OILS
Mustard
Oilseed rape
Canola
Sunflower
CEREALS
Wheat, Rice
Maize, Rye
LEGUMES
Soybean, Pigeon pea,
Chick pea

69. Ministry of Science and Technology
Department of Biotechnology
• Department of Science And Technology
• Department of Scientific and Industrial Research (Council for
Scientific and Industrial Research)
Ministry of Agriculture
• Department of Agricultural Research and Education
(Indian Council of Agricultural Research and Education)
Agencies of Public sector promotingAgencies of Public sector promoting
Agricultural BiotechnologyAgricultural Biotechnology
Agencies of Public sector promotingAgencies of Public sector promoting
Agricultural BiotechnologyAgricultural Biotechnology

70. Biotech Industry in India (2004-05)
Segment Revenues ($ million) Market Share (%) Growth
(%)2003-04 2004-05 2003-04 2004-05
BioPharma
BioServices
BioAgri
BioIndustrial
Bioinformatics
625.45
62.50
29.55
54.09
18.18
811.36
96.59
75.00
72.73
22.73
79.19
7.91
3.74
6.85
2.30
75.24
8.96
6.95
6.74
2.11
29.72
54.55
153.85
34.45
25.00
Total Industry
Size
789.77 1078.41 100.00 100.00 36.55
BioPharma corners three-fourth of Indian market ($811 million out of
$1070 million)

71. Transgenic research in India (Public Sector)
 AAU, Jorhat, Assam
 Bose Institute, Kolkata
 Central Institute for Cotton Research, Nagpur
 Central Potato Research Institute, Shimla
 Central Tobacco Research Institute, Rajahmundry
 Centre for Cellular and Molecular Biology, Hyderabad
 Central Rice Research Institute, Cuttack
 Delhi University, South Campus, New Delhi
 Directorate of Rice Research, Hyderabad
 IARI, New Delhi
 IARI sub-station, Shillong

72. Transgenic research in India (Public Sector)
 International Centre for Genetic Engineering and
Biotechnology, New Delhi
 International Crop Research Institute for Semi-arid
Tropics, Hyderabad
 Indian Institute of Horticulture Research, Bangalore
 Jawaharlal Nehru University, New Delhi
 Madurai Kamraj University, Madurai
 Narendra Dev University of Agriculture, Faizabad
 National Botanical Research Institute, Lucknow
 Punjab Agricultural University, Ludhiana
 Tamil Nadu Agricultural University, Coimbatore
 TERI, New Delhi
 University of Agricultural Sciences, Bangalore

73. Transgenic research in India (Pvt. Sector)
 Ankur Seeds Limited, Nagpur
 Hybrid Rice International, Gurgaon
 Indo American Hybrid Seeds, Bangalore
 M/s MAHYCO, Mumbai
 Metahelix Life Sciences, Bangalore
 MAHYCO Research Foundation, Hyderabad
 Monsanto, Mumbai
 M/s Proagro PGS (India) Ltd., Gurgaon
 Syngenta India, Limited, Pune
 Sungro Seeds Ltd, New Delhi

74.  Cereals Rice, Wheat, Maize
 Grain legumes Chickpea, Mungbean,
Black gram, Pigeonpea
 Oilseeds Mustard, Ground nut
 Vegetables Brinjal, Tomato, Potato,
Chilli, Cabbage, Cauliflower
 Fruits Papaya, Banana, Muskmelon
 Medicinal plants Brahmi
 Others Cotton, Coffee, Tobacco
Target crops for transgenic research
in India

75. Target traits
 Disease resistance
 Improving the quantity of the protein
 Increasing vitamin content
 Stress tolerance
 Herbicide resistance
 Delayed ripening
 Edible vaccine

76. Transgenic crops approved for field trials in 2005
Brinjal
 Mahyco, Mumbai cry1Ac
 Sungro Seeds Ltd, New Delhi cry1Ac
 IARI, New Delhi cry1F
Cabbage
 Sungro Seeds Ltd, New Delhi cry1Ac
Cauliflower
 Sungro Seeds Ltd, New Delhi cry1Ac
Corn
 Monsanto, Mumbai Cry1Ab
 Metahelix Life Sciences, Bangalore Modified Mu-element
(Turbo-Mu)

77. Transgenic crops approved for field trials in 2005
Cotton
 Ajeet Seeds, Aurangabad cry1Ac, cryX
 Ankur Seeds P.Ltd., Nagpur cry1Ac, cryX
 M/s Bioseed Research India Pvt Ltd, Hyd cry1Ac, cryX
 M/s Emergent Genetics India P. Ltd, Hyd cry1Ac, cryX
 Ganga Kaveri Seeds Ltd, Hyderabad cry1Ac
 Green Gold Seeds Ltd, Aurangabad GFM cry1Aa
 JK Agri Genetics, Hyderabad cry1Ac
 M/s Kaveri Seeds Co. P. Ltd, S’bad cry1Ac
 Krishidhan Seeds, Jalna cry1Ac, cryX
 Mahyco, Mumbai cryX
 Metahelix Life Sciences, Bangalore cry1Ac
 Nandi Seeds Pvt. Ltd Mehbubnagar cry1Ac
 Namdhari Seeds Pvt. Ltd, Bangalore cry1Ac

78. Transgenic crops approved for field trials in 2005
Cotton
 Nath Seeds, Aurangabad GFM cry1Aa
 Nuziveedu Seeds, Hyderabad cry1Ac, cryX
 Prabhat Agri Biotech Ltd. Hyderabad cry1Ac
 Pravardhan Seeds Pvt. Ltd Hyderabad cry1Ac
 Proagro Seeds Co. Ltd Hyderabad cry1Ac
 Rasi Seeds Ltd., Attur cryX
 Syngenta India Ltd., Pune Vip-3A
 Tulsi Seeds, Guntur cry1Ac, cryX
 UAS, Dharwad cry1Ac
 Vibha Agrotech Ltd. Hyderabad cry1Ac
 Vikki’s Agrotech, Hyderabad cry1Ac
 Vikram Seeds Ltd, Ahmedabad cry1Ac
 Zuari Seeds Ltd. Bangalore GFM cry1Aa

79. Transgenic crops approved for field trials in 2005
Groundnut
 ICRISAT, Hyderabad Coat protein of IPCV,
Nucleo Capsid Protein
of PBNV
Mustard
 UDSC, New Delhi barnase & barstar
Okra
 Mahyco, Mumbai cry1Ac
Pigeonpea
 ICRISAT, Hyderabad cry1Ac
 IARI, New Delhi cry1Ac, cry1Aa + cry1B
 Mahyco, Mumbai cry1Ac
 Metahelix Life Sciences, Bangalore NHX gene
Tomato
 IARI, New Delhi anti-sense replicase gene
of tomato leaf curl virus
 Mahyco, Mumbai cry1Ac

80. Bt cotton in India
Year Area under cultivation
(ha)
2002 50,000
2003 100,000
2004 5,00,000
2005 13,00,000

81. Bt cotton producing states in India
State Area (M ha)
 Maharashtra 0.607
 Andhra Pradesh 0.283
 Gujarat 0.164
 Madhya Pradesh 0.148
 Karnataka 0.040
 Tamil Nadu 0.029
 Northern states 0.064
Total 1.335

82. S. No Name of Hybrid Name of Company Zone
1 NCS – 207 Mallika M/s Nuziveedu Seeds Ltd. Central & South
2 NCS – 145 Bunny M/s Nuziveedu Seeds Ltd. Central & South
3 RCH 2 Bt M/s Rasi Seeds Ltd Central & South
4 RCH –144 Bt M/s Rasi Seeds Ltd Central
5 RCH –118 Bt M/s Rasi Seeds Ltd Central
6 RCH - 138 Bt M/s Rasi Seeds Ltd Central
7 RCH – 20 Bt M/s Rasi Seeds Ltd South
8 RCH – 368 Bt M/s Rasi Seeds Ltd South
9 RCH – 134 Bt M/s Rasi Seeds Ltd North
10 RCH – 317 Bt M/s Rasi Seeds Ltd North
Bt cotton varieties approved for commercial
cultivation in various zones
Source : MoEF

83. Bt cotton varieties approved for commercial
cultivation in various zones
S. No Name of Hybrid Name of Company Zone
11 MRC – 6322 Bt M/s Mahyco South
12 MRC – 6918 Bt M/s Mahyco South
13 MRC – 6301 Bt M/s Mahyco Central & North
14 MRC – 6304 Bt M/s Mahyco North
15 Ankur – 651 Bt M/s Ankur Seeds Ltd Central & North
16 Ankur 2534 Bt M/s Ankur Seeds Ltd North
17 Ankur – 09 M/s Ankur Seeds Ltd Central
18 MECH 12 Bt* M/s Mahyco Central (renewed)
19 MECH 162 Bt* M/s Mahyco Central & South (renewed)
20 MECH 184 Bt* M/s Mahyco Central & South (renewed)
Central zone : MP, Maharashtra & Gujarat * not renewed for AP
South Zone : AP, Karnataka & Tamil Nadu
North Zone : Punjab, Rajasthan & Haryana

84. Bt cotton varieties approved for large-
scale trials during Kharif 2005
Central Zone
1. VCH-111 Bt : M/s Vikki Agrotech Ltd
2. GK- 204 Bt and GK- 205 Bt : M/s Ganga Kaveri Seeds
3. PRCH-102 Bt : M/s. Paravardhan Seeds Pvt. Ltd
4. NPH-2171 : M/s Prabhat Agri Biotech Ltd
5. RCH 377 Bt and RCH – 386 Bt : M/s Rasi Seeds Ltd
6. ACH-155-1 : M/s Ajeet Seeds
7. Tulasi 4 Bt & Tulasi –117 Bt : M/s Tulasi Seeds Pvt. Ltd
8. Brahma Bt : M/s Emergent Genetics India Pvt. Ltd
9. NCS 913 Bt : M/s. Nuziveedu Seed Ltd

85. Bt cotton varieties approved for large-scale
trials during Kharif 2005
Central Zone
10. KDCHH - 9810 Bt, KDCHH-621 BG - II, KDCHH-441
BG- II : M/s Krishidhan seeds Ltd
11. RCH 515 BGII : M/s Rasi Seeds Ltd
12. MRC- 7301 BG II, MRC-7326 BG II, MRC-7351 BG II,
MRC-7341 BG II and MRC-7347 BG II : M/s Mahyco,
13. JK-Varun and JKCH 99 Bt : M/s J.K. Seeds
14. NCEH-2R Bt : M/s. Nath Seeds Ltd
15. 02- 41 Vip : M/s Syngenta Seeds India Ltd
16. ACH –11-2 BG-II and ACH –155-2 BG –II : M/s Ajeet
Seeds Ltd.
17. VICH 5 and VICH 9 : M/s Vikram Seeds

86. Bt cotton varieties approved for large-scale
trials during Kharif 2005
South Zone
1. GK-207 Bt and GK-209 Bt : M/s Ganga Kaveri Seeds
2. NPH-2270 Bt and NPH-2171 Bt : M/s Prabhat Agri
Biotech Ltd
3. PRCH-102 Bt and PRCH-103 Bt : M/s Paravardhan
Seeds Pvt. Ltd
4. KDCHH-9632 Bt and KDCHH-9810 Bt : M/s Krishidhan
Seeds Ltd
5. RCHB-708 Bt, RCHB-111 Bt and RCHB-371 Bt : M/s Rasi
Seeds Ltd
6. NCS-913 Bt : M/s Nuziveedu Seeds Ltd
7. Brahma Bt and Paras Laxmi : M/s Emergent Genetics
India Pvt. Ltd

87. Bt varieties approved for large-scale trials
during Kharif 2005
South Zone
8. MRC-7160 BG-II, MRC-7201 BG-II, MRC-7351 BG-II &
MRC-7347 BG-II : M/s Mahyco
9. RCH-530 BG-II & RCH-533 BG-II : M/s Rasi Seeds Ltd
10. KDCHH-621 BG-II : M/s Krishidhan Seeds Ltd
11. J.K. Durga Bt & JKCH-99 Bt : M/s J.K. Seeds Ltd
12. NCEH-3R Bt : M/s Nath Seeds Ltd
13. Bunny Vip (02-41 Vip) & 02-42 Vip : M/s Syngenta
Seeds Ltd
14. VICH 5 & VICH 9 : M/s Vikram Seeds

88. Bt varieties approved for large-scale trials
during Kharif 2005
North Zone
1. VICH 9 : M/s Vikram Seeds
2. MRC 6025 BG-I, MRC 6029 BG-I : M/s Mahyco
3. RCH 314 Bt & RCH 308 Bt : M/s Rasi Seeds Ltd.
4. NCS 138 Bt and NCS 913 Bt : M/s Nuziveedu Seeds Ltd.
5. MRC 7017 BG-II and MRC 7031 BG-II : M/s Mahyco
6. JHCH 1947 Bt : M/s J.K Agri Seeds Ltd.
7. NECH 6 R Bt : M/s Nath Seeds Ltd.
8. 02-58 vip-3 : M/s Syngenta Seeds India Ltd.

91. Daffodils
Erwinia bacteria
Genes
Plasmids Agrobacteria
Kernel Hull
Embryo
Provitamin A
producing rice
embryo
1
2 3 4
Locally
important varieties

95. ß-carotene rich Mustard oil

96. Wide Hybridization
 TCT provides solution to overcome the barriers of distant
hybridization (Inter-specific and inter-generic)
 Pre and post zygotic barriers to hybridization can be
overcome using in-vitro fertilization and embryo, ovule or
pod culture respectively.
Examples:
(1) Development of inter-generic and inter-specific crosses
have been successfully obtained in tobacco, clover, corn,
rice, cole, canola, poppy and cotton.

97. (2) Shortening of breeding cycle in banana, rose and orchids
through embryo culture techniques.
(3) Development of Inter-specific and inter-generic hybrids
of a number of agriculturally important crops like cotton,
barley, tomato, rice, jute, Hordeum x Secale and
Triticum x Secale are some of important examples.
(4) Mingo, in particular, was a breakthrough, as it was the
first barley cultivar produced by using ‘Bulbosum
Techniques’.

98. Achievements

99. ROLE OF WIDE CROSSES IN CROP
IMPROVEMENT
Wide crosses are generally used to improve crop varieties for disease
resistance, pest resistance, stress resistance, quality, adaptation, yield etc.
These crosses can even be used to develop new crop species. Techniques like
alien addition and alien
substitution may also be effective.
IMPROVING THE CROP PLANTS FOR
a). Disease and insect resistance

100. b). Improvement in quality
c). Improvement in yield: This also been achieved through the use of wild Spp. in
some crops e.g. Oat, Vigna, Arachis, Potato, Tobacco.

101. Synthetic Seeds
1. Synthetic seed
technology:
encapsulation of
somatic embryos
covered in a protecting
gel.
2. Seedless fruits: plants
regenerated from
triploid endosperm are
unable to undergo
meiosis

102. Pathogen Eradication
 Tissue culture techniques
can be used for
production of virus free
plants through ‘Meristem
Culture’.
 Pisum sativum, Trifolium
repens and Citrus sp.
virus free plants have
been regenerated using
shoot meristem culture.
 Downy mildew resistance
in bajra, leaf blight
resistance in potato,
chlorosis resistance in
tobacco.

103. Conclusion
Tissue culture will continue to play a key role in
the genetic engineering process for the foreseeable
future, especially in efficient gene transfer and
transgenic plant recovery.
Further, a country like India where the populations
are increasing at alarming rate, size of cultivable
lands are decreasing, major crops are witnessing
the yield plateau, TCT provides great hope not
only to create new combinations of genes but also
to ensure the quantity and quality of various food
commodities.

104. Thanks