This document discusses plant tissue culture and micropropagation techniques. It begins with an introduction to plant tissue culture and defines it as the growth of plant cells, tissues or organs in a sterile nutrient culture medium under controlled conditions. The document then provides a brief history of plant tissue culture, outlines the process of micropropagation including initiation, multiplication, rooting and acclimatization stages. It discusses using micropropagation to produce disease-free plants by culturing apical meristems and provides examples of various plants that have been made disease-free through this method. The advantages and disadvantages of micropropagation are also summarized along with current applications and future prospects of plant tissue culture techniques.

1. Title and Content Layout with List
First level
Second level
Third level
Fourth level
Fifth level
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2. Developing disease free plant stock in tissue
culture
Muhammad Usman Mughal
Roll no 25
M.Sc. Botany 3rd Semester
21 February 2015

3.  contents
 Introduction
 What is plant tissue culture?
 Brief history of Plant tissue culture
 Production of disease free plants
 Elimination of SCYLV from sugarcane plants
 A brief list of disease free plants
 Advantages of micropropagation
 Disadvantages of micropropagation
 Current and future status of plant tissue culture
 Conclusion
 References 21 February 2015 3

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5.  What is plant tissue culture?
 Growth of cells from a tissue
 Asexual propagation
 Under laboratory conditions
 Nutrient culture medium
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6.  Brief history of tissue culture
• - 1902 - Haberlandt proposed concept of in vitro cell
culture
• -1922 - Kolte and Robbins successfully cultured root and
stem tips respectively
• - 1946 - Ball raised whole plants of Lupinus by shoot tip
culture
• - 1954 - Muir was first to break callus tissues into single
cells
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7.  - 1957 - Skoog and Miller gave concept of hormonal
control (auxin: cytokinin) of organ formation
 - 1962 - Murashige and Skoog developed MS medium
with higher salt concentration
 - 2005 - Rice genome sequenced under International Rice
Genome Sequencing Project
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8.  Production of disease free plant
Systematically infected with one or more virus pathogen
Most of plant are infected by fungi, virus & bacteria
No commercially treatment to cure virus infected plants
Micropropagation provides a rapid method for production
of plants
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9.  Micropropagation of apical meristem
Application of plant tissue culture technique to clone
species using small pieces of mother cell
For developing disease free plant only cells of apical
meristem or axillary bud used
Rate of cell division at meristem
higher as compared to the division
of viruses
In this region no vascular bundles
present so viruses not move to that
region
21 February 2015 9http://www.uic.edu/classes/bios/bios100/labs/meristem.jpg

10.  Laboratory for tissue culture must be organized
 Use glassware that has only been used for plant tissue
culture
 Used only high purity water in plant tissue culture
 Plant must be healthy and actively growing
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11.  Aseptic Techniques
Technique Material sterilize
Steam sterilization/Autoclaving
(121°C at 15 psi for 20-40 min)
Nutrient media, culture vesels, glasswares and
plasticwares
Dry heat (160-180°C for 3h) Instruments (scalpel, forceps, needles etc.),
glassware, pipettes, tips and other plasticwares
Flame sterilization Instruments (scalpel, forceps, needles etc.),
mouth of culture vessel
Filter sterilization (membrane filter
made of cellulose nitrate or cellulose acetate of
0.45- 0.22μm pore size)
Thermolabile substances like growth factors,
amino acids, vitamins and enzymes.
Alcohol sterilization Worker’s hands, laminar flow cabinet
Surface sterilization (Sodium hypochlorite,
hydrogen peroxide, mercuric chloride etc)
Explants
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Rai, R., Campus, P., & Conservation, G. (1990). GENETICS AND PLANT BREEDING, p 10

12.  Components of medium
 Inorganic nutrients (N2,P,Ca,Mg,S)
 Carbon source (sugar)
 Organic supplements including
Vitamins (Thiamine, nicotinic acid, panthonic acid,
pyridoxine)
Amino acids (L-glutamine, L-asparagine, L-cysteine, L-
glycine)
Complex organics (casein hydrolysate, coconut milk, yeast
extract, orange juice, tomato juice)
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13.  Plant growth harmones
Auxins (root)
Cytokinins (shoot)
Gibbrellins (internode elongation, meristem growth)
Abscissic acid (for culturing woody species)
 Solidifying agent (agarose)
 pH (optimum is 5.8) lower than 4.5 or higher than 7.5
greatly inhibit the growth
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14.  Elimination of SCYLV from infected sugarcane
plants
 In the late 1980s, sugarcane yellow
leaf syndrome (YLS) was reported in
Hawaii, Australia and Brazil.
 During the 1990s, it was also detected in Florida and
Louisiana. Symptoms of YLS consist
of a leaf yellowing appearing first in
the midrib and leaf tip from where it
spreads downward, eventually
resulting in total leaf chlorosis.
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Hussain, A., Ahmed, I., Qarshi, Nazir, H. and Ullah, I. 2012. Plant Tissue Culture: Current Status and Opportunities. P 10.
Stage 0: preparation
of donor plant
Stage 1:
Initiation stage
Stage 2:
Multiplication stage
Stage 3:
Rooting stage
Stage 4:
Acclimatization stage

16. Photographs of sugarcane development from bud meristem explant to regenerated plant: (a) freshly excised bud
meristem; (b) after 2 weeks, the meristem just emerging surrounded by leaf scales, which turned brownish; (c) after 6
weeks, with embryogenic calli in the middle and nonembryogenic calli at the sides; (d) embryogenic callus ready for
regeneration; (e) after 2 months, regenerated plants in a Petri dish; (f) after 4 months, regenerated plants in soil
Fitch, M., Lehrer, A., Komor, E., & Moore, P. (2001). Elimination of Sugarcane yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by tissue
blot immunoassay. Plant Pathology, 50(6), 678.
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17.  A brief list of disease free plants
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Sr# Plant species Virus eliminated References
1 Brasica oleracea
(cauliflower)
CbSvr
TuMV,CIMV
Paludan (1971)
Walkey et. al. (1974)
2 Fragaria sp
(strawberry)
Crinckle
Yellow virus complex
Kacharmozov and izovorsaka
(1974)
Miller and blekengren (1963)
3 Malus sp
(apple)
Latent virus Campbell (1962)
4 Musa sp
(banana)
CMV, unidentified Berg and bustamanate (1974)
5 Nicotiana tobacum TMV
Dark green island of TMV
White et. Al (1977)
Murakishi and Carlson (1976)
6 Rubus ideaus
(rusberry)
Mosaic Putz (1971)
7 Saccharum officinarum
(sugercane)
SCYLV
Mosaic
Fitch et. Al (2001)
Raj et. Al (1991)
8 Solanum tuberosum
(Potato)
PaVM
PSTV
PVG
Dhingra et. al (1982)
Lizarraga et. al (1980)
9 Vitis vinifera GFLV
AMV
Monette (1986)
Monette (1986)
10 Zingiber officinale Mosaic Wang and Hu (1980)

18.  Advantages of micropropagation
 Producing disease free plant
 High fecundity rate , producing thousands of propagules
 Some plants with very small seeds including orchids are
growing through micropropagation
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 Disadvantages of micropropagation
• It is very expensive and can have a labor cost more than 70%
• Some plants are very difficult to disinfect of fungal organism
• Not all plants can be successful cultured due to proper medium,
for growth, is not known

19.  Current and future status of Plant tissue culture
 The past two decades of plant cell biotechnology has
evolved as a new era in the field of biotechnology,
focusing on the production of a large number of secondary
plant products
 The number of farmers who have incorporated transgenic
plants into their production systems in 2008 was 13.3
million, in comparison to 11 million in 2007.
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20.  University of the Punjab, pakistan
• Department of Botany
i. In “Plant Biotechnology Research Laboratory”
researcher developing disease free plant stock in tissue
culture under supervision of Dr. Humaira Afrasiab. Plants
including Garlic (Allium sativum), Grape (Vitis vinifera),
Amaryllis sp, Rice (Oryza sativa) and Lemon grass
(Cymbopogon sp).
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21. ii. In “Developmental and Regenerative Biology
Laboratory” researcher developing disease free plant stock
in tissue culture under supervision of Prof. Dr. Faheem
Aftab. Plants including Teak (Tectona grandis), Potato
(Solanum tuberosum L.), Pinus sp, Jojoba (Simmondsia
chinensis) and Jatropha curcas.
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22. • School of Biological Sciences (SBS)
Dr. Javed Iqbal, Professor Emeritus, Director of School of
Biological Sciences worked on Transformation and tissue
culture of Brassica napus, Gossypium sp, Oryza sativa L.,
Citrus reticulata L. Triticum aestivum L, Cicer arietinum L
and other cultivated plants.
 Center of Applied Molecular Biology (CAMB)
Dr. Tayyab Husnain Professor & Director of CAMB
worked on Oryza sativa L. Gossypium hirsutum L.,
chickpea plant, Sugarcane and still working on other
cultivated plants.
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23.  National Agricultural Research Centre (NARC)
National Agricultural Research Centre (NARC), Islamabad,
established in 1984, is the largest research center of the
Pakistan Agricultural Research Council (PARC). Under the
supervision of Dr. M. Azeem Khan, Director General of
NARC, working on Okra, Bottle gourd, Bitter gourd,
Sponge gourd, Vegetable Marrow, Cucumber, Radish,
Turnip, Carrot, Spinach,Tomato, Chili, Eggplant, Cabbage,
Lettuce, Onion and different English vegetables
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24.  National Institute for Genomics and Advanced Biotechnology (NIGAB)
 Tissue Culture Laboratory established at NARC in 1982 is known
to be the pioneer tissue culture facility in the country with
emphasis on pre-basic virus-free potato seed and producing
clones of other crops like, date palm, rice, carnation and banana.
The laboratory earned a name in production of disease-free potato
seed and banana plants.
 Under the supervision of Dr. Ghulam Muhammad Ali, important
cultivated plants including tomato (Lycopersicon esculentum),
Pakistani peanut (Arachis hypogea) and Pakistani Wheat
(Triticum aestivum) developed disease free plants and still
working on other cultivated plants.
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25.  Assiut University, Egypt
•Department of Vegetables, Faculty of Agriculture
 Prof. Dr. Azza Abdel-Aziz Ali Tawfik, produced disese free plant including
Gerbera sp, Rose (Rosa sp), Carnation (Dianthus caryophyllus), Rhododendron sp,
Anthurium sp, Rosemary (Rosmarinus officinalis), Salvia sp (sage), Eucalyptus sp,
Melaleuca sp and further working on ornamental and medicinal plants.
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27.  references
 Rai, R., Campus, P., & Conservation, G. (1990). GENETICS AND PLANT BREEDING.
 Hussain, A., Ahmed, I., Nazir, H., & Ullah, I. (2012). Plant tissue culture: Current
status and opportunities. Recent advances in plant in vitro culture, 1-28.
 Hussain, A., Ahmed, I., Qarshi, Nazir, H. and Ullah, I. 2012. Plant Tissue Culture:
Current Status and Opportunities
 Bhojwani, S. S., & Razdan, M. K. (1986). Plant tissue culture: theory and practice:
Elsevier.
 Rai, R., Campus, P., & Conservation, G. (1990). GENETICS AND PLANT BREEDING.
 Fitch, M., Lehrer, A., Komor, E., & Moore, P. (2001). Elimination of Sugarcane
yellow leaf virus from infected sugarcane plants by meristem tip culture visualized by
tissue blot immunoassay. Plant Pathology, 50(6), 676-680.
 Prammanee, S., Thumjamras, S., Chiemsombat, P., & Pipattanawong, N. (2011).
Efficient shoot regeneration from direct apica meristem tissue to produce virus-free
purple passion fruit plants. Crop Protection, 30(11), 1425-1429.
 Rout, G. R., Mohapatra, A. and Jain, M. S. 2006. Tissue culture of ornamental pot
plant: A critical review on present scenario and future prospects, Science Direct, 24:
531–560
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28.  Kothari, S., Joshi, A., Kachhwaha, S., & Ochoa-Alejo, N. (2010).
Chilli peppers—a review on tissue culture and transgenesis.
Biotechnology advances, 28(1), 35-48.
 Cheema, K. L., & Hussain, M. (2004). Micropropagation of
sugarcane through apical bud and axillary bud. Int J Agri Biol, 6, 257-
259.
 Jain, S. M. (2001). Tissue culture-derived variation in crop
improvement. Euphytica, 118(2), 153-166.
 Bhatia, P., Ashwath, N., Senaratna, T., & Midmore, D. (2004). Tissue
culture studies of tomato (Lycopersicon esculentum). Plant Cell, Tissue
and Organ Culture, 78(1), 1-21.
 Debnath, M., Malik, C., & Bisen, P. (2006). Micropropagation: a tool
for the production of high quality plant-based medicines. Current
pharmaceutical biotechnology, 7(1), 33-49.
 Jaskani, M. J., Abbas, H., Khan, M., Qasim, M., & Khan, I. (2008).
Effect of growth hormones on micropropagation of Vitis vinifera L. cv.
Perlette. Pakistan Journal of Botany, 40(1), 105.
21 February 2015 28

29.  Jahangir, G. Z., Nasir, I. A., Sial, R. A., Javid, M. A., & Husnain, T. (2010). Various
Hormonal Supplementations Activate Sugarcane Regeneration In-Vitro. Journal of
Agricultural Science, 2(4), p231.
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callus senescence and necrosis in chickpea (Cicer arietinum L.) Pakphyton 3:119-125.
 Iqbal, J. and Azam, N. 1991. Changes in protein content during in vitro callogenesis
and embryogenesis in Citrus reticulata L. cv. Blanco. III. J. Syst. & Exp. Biol. 1:44-507-251.
 Rashid, B., Husnain,T. and Riazuddin, S. (2009). Rapid in vitro root induction in
transgenic cotton shoots. Plant Tissue Cult. & Biotech. 19(2): 24
 Majeed, A, Husnain, T. and Riazuddin, S. (2000). Transformation of Virus Resistant
Genotype of Gossypium hirsutum L., with Pesticidal Gene. Plant Biotech 17(2): 105-110.
 Khanum, F., Husnain, T. and Riazuddin, S. (1998). Effect of age of seedling and
phytohormones on micropropagation of indica rice (Oryza sativa L.) from meristem culture.
J. Plant Biol. 41(2): 93-96.).
 Afroz, A., Chaudhry, Z., Rashid, U., Khan, M. R., & Ali, G. M. (2010). Enhanced
regeneration in explants of tomato (Lycopersicon esculentum L.) with the treatment of
coconut water. African Journal of Biotechnology, 9(24), 3634-3644.
 Hassan, M., Akram, Z., Ajmal, S., Mukhtar, T., Nasim, S., Shabbir, G., et al. (2013).
Highly efficient in vitro root induction in peanut by mechanical stress method. JAPS,
Journal of Animal and Plant Sciences, 23(2), 425-429
21 February 2015 29

30.  Rashid, U., Ali, S., Ali, G. M., Ayub, N., & Masood, M. S. (2009).
Establishment of an efficient callus induction and plant regeneration system in
Pakistani wheat (Triticum aestivum) cultivars. Electronic Journal of Biotechnology,
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