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21 February 2015 1

Developing disease free plant stock in tissue culture

Muhammad Usman Mughal

Roll no 25

M.Sc. Botany 3rd Semester

21 February 2015

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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What is plant tissue culture?

Growth of cells from a tissue

Asexual propagation

Under laboratory conditions

Nutrient culture medium

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http://agritech.tnau.ac.in/bio-tech/biotech_tc_jainirrigation_clip_image002_0000.jpg

http://oxorchids.com.tw/cindex.files/oximg-smallsize/dsc11111.jpg

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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- 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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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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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

region21 February 2015 9http://www.uic.edu/classes/bios/bios100/labs/meristem.jpg

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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Aseptic TechniquesTechnique 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

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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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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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.21 February 2015 14

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http://www.ctahr.hawaii.edu/MBBE/Images/images/zhu/2%20YLSsymptm.JPG

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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

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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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)

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

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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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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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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• 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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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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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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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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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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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.

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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.

Iqbal, J., Haroon, M. and Ahmad, M.S. 1991. Acid Phosphatase: A possible marker of 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 callogenesisand 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

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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, 12(3), 4-5.

Smith, M., & Drew, R. (1990). Current applications of tissue culture in plant propagation and improvement. Functional Plant Biology, 17(3), 267-289.

http://www.aun.edu.eg/distance/agriculture/azza/principles_and_botanical_basis_o.htm

http://en.wikipedia.org/wiki/Plant_tissue_culture#Applications

http://en.wikipedia.org/wiki/Micropropagation#Disadvantages

http://en.wikipedia.org/wiki/Micropropagation#Advantages

http://agriinfo.in/default.aspx?page=topic&superid=3&topicid=1884

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http://www.aun.edu.eg/membercv.php?M_ID=1191

http://www.parc.gov.pk/index.php/en/nigab

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http://www.parc.gov.pk/index.php/en/2013-04-11-06-13-50/narc-islamabad

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