n.z nyawo

Biotechnology in the
Making
Plant Technology

Science in Action, a
breath away from
victory
A scientist who is only a
scientist is not a scientist -
unknown
Who AM I
About Me
Student: Nosisa Zuziwe Nyawo
Full Time - Contact
Postgraduate
BSc Hons. Biological Science
Contact Me
Phone: 079 593 9030
Email: 213523195@stu.ukzn.ac.za
Web: www.ukzn.ac.za
UNIVERSITY OF
KWAZULU-NATAL
UNIVERSITY OF KWAZULU-NATAL
Office: John Bews
Block C, Room 108
School of Life Sciences

Therefore there has been an advancement in
various ways for culturing plant cells and
tissues. Indeed such capabilities have
contributed a lot in culturing of commercial
and pharmaceutical rare plants and their
associated chemicals.
Tissue Culture
One of the fascinating and controversial fields in our
days is Biotechnology. This concept developed in the
1970 after the development of genetic engineering
where scientists has been allowed to modify the
genetic material from a living cells. The manipulation
of the DNA molecules from plants, animals and many
more organisms is so called genetic engineering
Every cell has a DNA (double stranded molecule that
stored genetic information). Genes are passed from
parents to offspring but now scientists use the
Biotechnology concepts to culture cells.
The fact that secondary metabolites has
previously been produced even via laboratory.
Most plants undergo both primary and secondary
metabolism. Primary metabolites are directly used
for growth and development while secondary
metabolites are the end products of the primary
metabolites.
Secondary metabolites are produced by the plant
mature cells for different purposes e.g. defense.
This is the process whereby an explant (small pieces of
living tissue is isolated from an organisms and grown
aseptically in the nutrients media with the controlled
conditions. In vitro cultivation of plants is a necessary
step in a large amount of experiments:
Micropropagation, creation of virus free plants, and
genetic transformation and the most obtained results
from an invitro is the cell proliferation of the explant
into a mass of relatively undifferentiated tissues called
callus which lots of clustered cells. Most plants
undergo both primary and secondary metabolism.
Primary metabolites are directly used

Different types of cell differentiation
Hormones: The mostly used media is Murashige and Skoog,
1962. Its contain hormones like cytokinin, auxin, and etc.

Plant biotechnology is now used in our daily
basis. Tissue culture and in vitro is now used to
produce secondary metabolites.

Major secondary metabolites

Culture types
• The use of plant cell cultures has overcome
several inconveniences for the production of
these secondary metabolites in purpose of
novel medicine.
• Most of the research efforts that use
differentiated cultures instead of cell
suspension cultures have focused on
transformed (hairy) roots. Agrobacterium
rhizogenes causes hairy root disease in plants.

Callus
This term refers to a
mass of undifferentiated
cells induced by
hormone treatment.

Hairy Roots
• For mass production of high value secondary
metabolites (Agrobacterium rhizogenes). It is the new
route for large scale secondary metabolite production
because of their fast and growth, genetic and
biochemical stability.

Transformation with Agrobacterium rhizogenes

Suspension Culture

The very first secondary metabolite products cultured
• Cantharanthus roseus: Production of Shikonin

Shikonin used for:
• Antimicrobial Insecticidal Antitumor Cancer
• This pigment is found in the family Boraginaceae that includes
Lithospermum, Arnebia, Alkanna, Anchusa, Echium and Onosma.
• The growing demand for plant-based natural products has made this
group of compounds one of the enthralling targets for their in
vitro production.

Different methods of increasing the levels of
shikonin in plant cells such as:
• Selection of cell lines: selection of high
producing cells
• Optimization of culture conditions:
controlled environment
• Elicitation: collection of requirements
• Genetic transformation
• Metabolic engineering
• It was produced in in-vitro
using: successful
pharmaceuticals
• Shikonin produced from
1980,s and used as a
cosmetic and as a cancer
treatment.

Cantharanthus roseus (madagascar periwickle): Found shikonin as a lipstick pigment

Although it is expensive
to produce secondary
metabolites using
tissue culture and it’s
never been used since
it requires >200g of
leaves to produce a
yield of 1g of alkaloid.
BUT
After Shikonin, many
research followed to
produce different
products that can be
used as a
pharmaceutical used
and as novel medicines.

In 2010,
• Used both suspension and hairy roots cultures
using the plant Rauvolfia serpentina, they also
used Plant hybridization techniques (process of
combining different varieties or species of
plants to create a hybrid)

Callus and cell suspension culture of Rauvolfia serpentine: production secondary
metabolites

Alkaloids: nitrogen containing base that are currently in clinical
Anti-neoplastic agents’ vinblastine and vincristine
Analgesics morphine and codeine
Anti-hypertensives ajmalicine
Serpentine anti-arrhythmic ajmaline

Hairy roots culture of
Rauwolfia serpentina

The hairy root culture of Rauwolfia serpentina (above) obtained by transformation with
Agrobacterium rhizogenes Produces indole alkaloids; it is capable of synthesizing 20
indole alkaloids, including 6 novel compounds.

The previous study of the fast-growing hairy root of Catharanthus roseus
has been selected for screening of indole alkaloid production. Ajmalicine,
serpentine, catharanthine and vindoline were identified by High
Performance Liquid Chromatography (HPLC) in intact roots of all clones.
Other large scale production secondary metabolites products in liters

In 2004, indole alkaloid
ajmalicine and serpentine
were produced using using
Murashige and Skoog
medium (MS) from the
mutant, Catharanthus
roseus plant. Cytokinin
increased the production of
ajmalicine, serpentine and
reserpine (high blood
pressure).
Above: Cartharanthus pusillus
and Below: Catharanthus roseus
produced Ajmalicine

Higher producer cell line in biotechnology is important
for the targeted substance to be more viable

Plant name Active ingredient Culture medium and plant growth regulator(s) Culture type Reference (s)
Aconitum heterophyllum Aconites MS + 2,4-D + Kin Hairy root Giri et al., 1997
Adhatoda vasica Vasine MS + BAP + IAA Shoot culture Shalaka and Sandhya, 2009
Agastache rugosa Rosmarinic acid MS + 2,4-D + Kin + 3% sucrose Hairy root Lee et al., 2007
Agave amaniensis Saponins MS + Kinetin Callus Andrijany et al., 1999
Ajuga reptans Phytoecdysteroids Hairy root Matsumoto and Tanaka, 1991
Aloe saponaria Glucosides MS + 2,4-D + Kinetin Suspension Yagi et al., 1983
Ambrosia tenuifolia Altamisine MS + Kinetin Callus Goleniowski and Trippi, 1999
Ammi majus Umbelliferone MS + BAP Shootlet Krolicka et al., 2006
Ammi visnaga Furanocoumarin MS + IAA + GA3 Suspension Kaul and Staba, 1967
Amsonia elliptica Indole alkaloids Hairy root Sauerwein et al., 1991
Anchusa officinalis Rosmarinic acid B 5 + 2,4-D Suspension De-Eknamkul and Ellis, 1985
Angelica gigas Deoursin MS (Liq.) + 2,4-D + GA3 Hairy root Xu et al., 2008
Anisodus luridus Tropane alkaloids MS + 2,4-D + BA Hairy root Jobanovic et al., 1991
Ammi majus Triterpenoid MS + 2,4-D + BA Suspension Staniszewska et al., 2003
Arachys hypogaea Resveratol G5 + 2,4-D + Kin. Hairy root Kim et al., 2008
Armoracia laphthifolia Fisicoccin MS + IAA Hairy root Babakov et al., 1995
Artemisia absinthum Essential oil MS + NAA + BAP Hairy root Nin et al., 1997
Artemisia annua Artemisinin MS + IAA + Kinetin Hairy root Rao et al., 1998
Artemisia annua Artemisinin MS + NAA + Kinetin Callus Baldi and Dixit, 2008
Aspidosperma ramiflorum Ramiflorin MS + 2,4-D + BAP Callus Olivira et al., 2001
Aspidosperma ramiflorum Ramiflorin alkaloid MS + 2-4,D + BAP + 30 g/l Sucrose Callus Olivira et al., 2001
Astragalus mongholicus Cycloartane saponin MS + 2,4-D + Kin Hairy root Ionkova et al., 1997
Astragalus mongholicus Cycloartane MS + IAA + NAA Hairy root Ionkova et al., 1997
Azadirachta indica Azadirachtin MS + 2,4-D Suspension Sujanya et al., 2008
Azadirachta indica Azadirachtin MS + 2,4-D + Cyanobacterial elicitor Suspension Poornasri Devi et al., 2008
Beeta vulgaris Betalain pigments MS + IAA Hairy root Taya et al., 1992
Brucea javanica Alkaloids MS + 2,4-D + Kinetin Suspension Lie et al., 1990
Brucea javanica Cathin MS + IAA + GA3 Suspension Wagiah et al., 2008
Brugmansia candida Tropane MS + 2,4-D + IAA Hairy root Marconi et al., 2008
Brugmansia candida Tropane alkaloid MS + BA + NAA Hairy root Giulietti et al., 1993
Bupleurum falcatum Saikosaponins B 5 + IBA Root Kusakari et al., 2000
Bupleurum falcatum Saikosaponins LS + 2,4-D Callus Wang and Huang, 1982
Calystegia sepium Cuscohygrine MS + 2,4-D + BA Hairy root Jung and Tepfer, 1987
Camellia chinensis Flavones MS + 2,4-D + NAA Callus Nikolaeva et al., 2009
Camellia sinensis Theamine MS + IBA + Kinetin Suspension Orihara and Furuya, 1990
Campanula medium Polyacetylenes MS + IAA + BA Hairy root Tada et al., 1996
Canavalia ensiformis Canavanine LS + NAA + Picloram Callus Ramirez et al., 1992
Capsicum annum Capsiacin MS + 2,4-D+ GA3 Callus Varindra et al., 2000
Capsicum annum Capsiacin MS + 2,4-D + Kin. Callus Umamaheswari and Lalitha, 2007
Capsicum annuum Capsaicin MS + 2,4-D + Kinetin Suspension Johnson et al., 1990
Table 1: The biotechnology
(tissue culture) and
potential products from
plant in recent years after
the discovery of the first
pigment Shikonin.

Cassia obtusifolia Anthraquinone MS + TDZ + IAA Hairy root Ko et al., 1995
Cassia senna Sennosides MS + NAA + Kin Callus Shrivastava et al., 2006
Catharanthus roseus Indole alkaloids MS + IAA Suspension Moreno et al., 1993
MS + NAA + Kinetin Suspension Zhao et al., 2001
Catharanthus roseus Vincristine MS + 2,4-D + GA3 Suspension Lee-Parsone and Rogce, 2006
Catharanthus roseus Indole alkaloid MS + 2,4-D + GA3 + Vanadium Suspension Tallevi and Dicosmo, 1988
Catharanthus roseus Catharathine MS + 2,4-D + UV-B radiation Suspension Ramani and Jayabaskaran, 2008
Catharanthus trichophyllus Indole alkaloids MS + IAA + GA3 Hairy root Davioud et al., 1989
Cayratia trifoliata Stilbenes MS + IAA + GA3 Suspension Roat and Ramawat, 2009
Centella asiatica Asiaticoside MS + 2,4-D Hairy root Kim et al., 2007
Centella asiatica Asiaticoside Ms + 2,4-D + Kin Callus Kiong et al., 2005
Centella asitica Asiaticoside MS + BAP + IAA Shoot Kim et al., 2004
Centella asitica Asiaticoside MS + 2,4-D Hairy root Paek et al., 1996
Centranthes ruber Valepotriates MS + IAA + Kin Hairy root Granicher et al., 1995
Cephaelis ipecacuanha Alkaloids MS + IAA Root Teshima et al., 1988
Chaenatis douglasei Thiarbrins MS + NAA Hairy root Constabel and Towers, 1988
Chrysanthemum
Pyrithrins MS + 2.4-D + Kinetin Callus Rajasekaran et al., 1991
cinerariaefolium
MS + Kinetin Suspension Kuch et al., 1985
Cinchona ledgeriana Quinine MS + 2,4-D Hairy root Hamill et al., 1989
B 5 + 2,4-D Suspension Schripsema et al., 1999
Citrus sp. Caffeine MS + 2,4-D + Kinetin Callus Waller et al., 1983
Coffea arabica Forskolin MS + IAA + Kin Hairy root Sasaki et al., 1998
Coleus forskohlii Corydaline MS + IAA + 3% sucrose Embryo Hiraoka et al., 2004
Corydalis ambigua Corydaline MS + IAA + GA3 Shoot Rueffer et al., 1994
Corydalis cava Alkaloids MS + 2,4-D + Kinetin Callus Iwasa and Takao, 1982
Corydalis ophiocarpa Corydalin MS + 2,4-D + BAP Callus Taha et al., 2008
Corydylis terminalis Berberin MS + 2,4-D + BAP Callus Khan et al., 2008
Coscinium fenustratum Berberine MS + IAA +BAP Callus Nair et al., 1992
Coscinium fenustratum Berberine MS + 2,4-D + GA3 Suspension Narasimhan and Nair, 2004
Coscinium fenustratum Flavonoid MS + 2,4-D + NAA + BAP Callus Maharik et al., 2009
Crataegus sinaica Plaunotol MS + NAA + BA Callus Morimoto and Murai, 1989
Croton sublyratus Anthraquinones LS + NAA + Kinetin Suspension Dornenburg and Knorr, 1996
Cryptolepis buchanani Essential oil MS + IAA + GA3 Shoot Quiala et al., 2006
Cymbopogon citratus Hyocyamine MS + IAA Hairy root Hilton and Rhodes, 1993
Digitalis purpurea Cardioactive glycosides MS + 2,4-D + BA Hairy root Saito et al., 1990
Diocorea doryophora Diogenin MS + 2,4-D + BA Suspension Huang et al., 1993
Drosera rotundifolia 7-Methyljuglone MS + BAP + NAA Shoot culture Hohtola et al., 2005
Duboisia leichhardtti Scopalamine MS + 2,4-D + BA Hairy root Muranaka et al., 1992
Echinacea purpurea Alkamides MS + 2,4-D Hairy root Trysteen et al., 1991
Eleutherococcus senticosus Eleuthrosides MS + 2,4-D Suspension Shohael et al., 2007
Ephedra sp. L-Ephedrine MS + Kinetin + 2,4-D Suspension O’Dowd et al., 1993
Eriobotrya japonica Triterpenes LS + NAA + BA Callus Taniguchi et al., 2002
Fabiana imbricata Rutin MS + NAA + 2,4-D Callus and
Schmeda-Hirschmann et al., 2004
Suspenson
Fagopyrum esculentum Flavonol MS + IAA + GA3 Hairy root Trotin et al., 1993
Fagopyrum esculentum Rutin MS + NAA Hairy root Lee et al., 2007
Frangula alnus Anthraquinones WPM + IAA + BAP Callus Kovacevic and Grabisic, 2005
Fritillaria unibracteata Alkaloids MS + 2,4-D + Kin Multiple shoot Gao et al., 2004
Gentiana macrophylla Glucoside MS + IAA + Kin Hairy root Tiwari et al., 2007
Gentiana sp. Glucosides B 5 + Kinetin Callus Skrzypczak et al., 1993

Biotechnology on biology
Shikonin: Anti-cancer
Ajmalicine: Heart attack
Quinine: Malaria prevention
Serpentine and reserpine : (Blood pressure)
Berberiney still depend : anti-inflammatory and anti-diabetic and reduce glucose production in the liver.
Ginsenosides Panax ginseng in 1997: traditional Chinese medicine

It is difficult to extracts compounds without using media:
Biotechnology sometimes is disadvantageous but its
most cases its:
MacQeen, H. 1998. Biotechnology puts the squeeze on plants. New Science 14, 50-54.