PLANT PROPAGATION (AGH 331)

II. VEGETATIVE PROPAGATION,

CLONE AND UNIFORMITY

Dr Darda Efendi
dardaefendi@yahoo.com
CP: 0812 88 635 027; WA/Line
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Vegetative Propagation Topics:

◼ Vegetative Propagation, Clone and

Uniformity
◼ Plant Propagation by Special Organs
(1)
◼ Plant propagation by Cutting (1)
◼ Plant Propagation by Layering (1)
◼ Budding and Grafting (combined
propagation) (1)
◼ Tissue Culture/Micro Propagation (4)

LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
Clone : Plant propagated through vegetative
propagation
Cultivar: Cultivated variety, Clone that has been
released and cultivated (potato, banana,
apple, stawberry)
Line : Plant population selected through seed
Variety: Line that has been released and cultivated
(rice, corn, chilly, tomato)

A clonal colony or genet is a group of genetically identical

individuals, grown in a given location, all originating vegetatively
from a single ancestor. An individual in a genet is referred to as
a ramet.
 The Reason for Using Clonal Propagation

1. To get superior propagule

2. Uniformity in a population
3. For helping propagation
4. Shorter juvenile phase
5. To combine more than one variety
in one individual plant
6. To control plant development
 The origin of Clonal Cultivars

◼ Historically, clonal population has been cultivated

since centuries. Naturally clone formation could take
million of years.
◼ Carbernet Sauvignon grape and Sultana have been
cultivated since 2000 years ago. Ambon and Raja
banana formed naturally since million years ago.
◼ Now? By using biotechnology, clone formation much
more faster (somatic hybrid, embryo-rescue; anther
culture; Genetic Transformation; Genome Editing)
 Apel
Domestication:
pear, More difficult
Potato cherry,
Sweet potato Easier plum
Bamboo Natural Apricot Grafting
Sugar cane vegetative
citrus
propagation
Banana
Technology: Hormone, media,
environmental control, tissue
Rambutan culture, syntetic seeds.
Durian
Layering
Litchi
Mango
ornamental,
Lansdscap,
Plantation plant
Forestry plant
 The origin of Clonal Cultivars

1. Seedling Selection
◼ Almost all of clonal cultivars originated from
selection of seedling population (without purposeful
or by planned breeding)
◼ P1 X P2 → S0 (ortet) → clonal population (ramets;
An individual member of a clone.)
◼ Clone must be test at multi locations
◼ Interaction Genotipe and environment →
phenotypic; P = G x E x GE
 ILUSTARATION OF SELECTION AND CLONAL MASS
PROPAGATION

P1 X P2 Parents generation

S0 Progeny Seedling
Vegetative Propagation

S1a S1b S1c S1d S1e S1z First clonal population

S2c Second clonal population

S3c Third clonal pupulation

Sxc X clonal generation

SxC=ortet= the original plant from which the members of a clone have descended
2. Mutation
◼ Mutation is the second source of cultivar
◼ Mutation occur on DNA and heritable (deletions,
duplications, translocations, inversions; aneuploidy,
polyploidy)
◼ Albino, variegated → example of mutation
◼ Apple Manalagi → mutation from Malang Apple (Rome
Beauty?)
Bud Mutation/Bud Sports/Mutasi Tunas

◼ Source of new varieties of fruits and other plants

LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
◼ Sport/bud sport (lusus): is a part of plant that shows morphological differences
from the rest of the plant.
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
 Induce Mutation: Physical (gamma ray); Chemical
(colchicines for polyploidy); Biological (genetic
transformation; Genome Editing)

CLONE

A. Natural Mutation B. Wild Type C. Gen. Transf.

V0 SN G0
cloning cloning
V1 Sn G1 cloning

Vx Gx
◼ Biotecknology: Developing induce mutation or changing
plant at selular and molecular level
◼ DNA Recombinant technology process include:
◼ isolation of gene target,
◼ engineer the gene,
◼ put the gene in E. coli for multiplication and storage,
◼ transfer the gene to plant nucleus by Agrobacterium or
gene gun,
◼ then regenerate the modified plant.
 Hin d III
Sph I (11083)
Pst I
Lac Z alpha
Sal I
Xba I
Bam H I CaMV35S promoter
Sma I (11052)
Kpn I
Sac I
Nco I
Eco R I Catalase intron
Bgl II
Spe I
Bst XI
GUSPlus
2x CaMV35S promoter
Nhe I (2026)
Xho I (10004) Pml I
Bst EII
hygromycin (R)
NOS polyA

T-BORDER (R)
Xho I (8910) pCAMBIA1305.1
CaMV35S polyA 11846 bp

T BORDER (L)

kanamycin (R) pVS1 Sta

pBR322 ori
pBR322 bom pVS1 rep Nhe I (5467)

Plasmid
 VARIATION in CLONE
1. Chimeras:
◼ Genetic diversity in one individual. Occur because
of single cell mutation on organ and structure that
inherited to next generation (exp. variegated leaf)
◼ Chimera: Periclinal, Mericlinal, sectoral
→ heritable
Chimera: when cells of more
than one genotype (genetic
makeup) are found growing
adjacent in the tissues of that
plant.

Variegated guava
graft chimera
 Origin of
Chimera
histogen
(L1: outside,
L2: middle
L3: inside)

Gymnospermae, root or young

embryo
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
3 type of chimera:
1. Periclinal
2. Mericlinal
3. Sectorial
http://generalhorticulture.tamu.edu/HORT604/LectureSuppl/AnatomyChimeras
/AnatomyChimeras05.htm
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
 2. Transposable element
◼ Transposon: (Transposable element, mobile genetic
element, Jumping DNA), found by Dr. Barbara
McClintock based on unique genetic diversity.
Symptom usually specific uniform color with spot
pattern and size (Colocasia, Diffenbachia)
 NON GENETIC VARIATION
Effect of Environment
(Interaction of Genetic and Environment)
(Temperature, Photoperiod, Light intensity)

Fruit size and form, plant age,

appearence, bloom ability, tuber,
tuber size, plant Vigor, juvenility
change, etc.
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
 NON GENETIC VARIATION

1. Effect of environment on phenotypic diversity

(teorytically, plants in one clone have the same
genetic make up, so selection based on
phenotypic is not recomended because
phenotypic is a fuction of genotype x
environment) ,
2. There are growth and maturity variation and
change (juvenility vs maturity)
 http://www.uky.edu/Ag/Horticulture/Geneve/teaching/plan
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
tpropwebpages/geneticselection/geneticphasechange.pdf
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
❑ Topophysis: Difference in growth orientation
(ortotrop: vertical; plagiotrop: horisontal→
climbing pepper from ortotrop shoot; shrubs
pepper from plagiotrop branch; Genetum
gnemon, ‘cemara buaya’)
❑ Sistemic patogen infection (virus, mycoplasma,
bacteria, fungi, viroid,)
a. Virus (PVY, PLRV, CaMV,
b. Phytoplasma (mycoplasma-like Organism)
(Aster Yellow pada strawbery)
c. Bakteri
d. Viroid (citrus exochordis, Avocado sunbloth,
Crysanthemum stunt)
LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA
 CLONING MANAGEMENT

◼ Characterization of source of propagule: to

guarentee the trueness-to-name → plant finger
print test with DNA analysis (Isozyme analysis,
RFLP – Restriction Fragment Length Polymorphism;
PCR – Polymerase Chain Reaction dan RAPD –
Randomly Amplified Polymorphic DNA)
◼ True-to-type clonal progenies test → Phenotypic
selection and then genotypic selection
DNA Finger Printing isoenzime
 CLONING MANAGEMENT

◼ Free from Patogen:

1. Visual inspection
2. culture indexing
3. Virus Indexing
4. serology test
5. Biochemical metod → electrophoresis
virus detection
PATOGEN INDEXING

Virus indexing on Verticillium

GRAFTING/BUDDING FOR VIRUS TRANSMISSION
INDEXING
Meristem
Culture
 METHOD FOR PATOGEN ELIMINATION

1. Selection of non-infected part of plant ( on plant

infected by fungi or bacteria)
2. Meristem culture ( on virus infected plant)
3. Heat treatment (40-60 oC)
4. Thermoteraphy (virus elimination) (40 oC, 7 days)
5. Propagated by seed (most virus are not transmitted
by seed)
 INDENTIFICATION OF VIRUS-FREE CLONE

Clone
virus identification
Sxa Sx b
V- V+

clone No.1
Heat treatment No Treatment
DISCARD
Foundation Block

Registration and Certification

Commercial Distribution
FOUNDATION CLONAL SYSTEM

1. Innitial selection
2. Maintenance in foundation block
3. multiplication and distribution
ILUSTRATION OF NURSERY MANAGEMENT

Development
Initial analysis
Visual Inspection
Nuclear
virus test
Stock
progeny test
Step 1. Development
Step 2. Maintenance Step 3. Multiplication and distribution

Source
Block
Foundation
Increase
or Propagation Block
Mother Block

Commercial
Plants
Salacca zalacca

LABORATORY OF BIOTECHNOLOGY
DEPARTMENT OF AGRONOMY
BOGOR AGRICULTURAL UNIVERSITY (IPB)
INDONESIA