morphogenesis in organogenesis and somatic embryogenesis...
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The Development and Impact of The Development and Impact of inin--vitrovitroMorphogenesis in Organogenesis and Somatic Morphogenesis in Organogenesis and Somatic Embryogenesis of Bananas (Embryogenesis of Bananas (MusaMusa spp.)spp.)
Department of Horticulture, National Taiwan University, Taipei, Taiwan
Department of Post-Modern Agriculture, MingDao University, ChungHua, Taiwan
Division of Forest Biology, COA Forestry Research Institute, Taipei, Taiwan
Council of Agriculture Executive Yuan, Taipei, Taiwan
J.P. Chung, I.C. Huang, M.C. Chung, Y.W. Liauh,P.L. Huang, and C.T. Shii
The biotechnology of plant tissue culture is helpful for agriculture, particularly, crucial for triploid banana crops in diverse fields.1. Applying in healthy, uniform, vigor, mass clonal propagation.2. Inducing mutagenesis and in vitro selection.3. Breeding of biotic and abiotic stress resistances.4. In vitro germplasm conservation of clonal repository.5. Molecular breeding of transgenic bananas.6. Biotechnology of biotransformation.
The subsequent application of plant tissue culture are based on in vitro morphogenesis categorized to two major pathways.
1. In vitro organogenesis -- Caulogenesis : ex. adventitious buds-- Rhizogenesis : ex. adventitious roots
2. In vitro somatic embryogenesis-- Asexual embryos-- Artificial seeds
Table 1. Comparisons of in vitro morphogenesis between organogenesis and somatic embryogenesis
Characters Organogenesis Somatic embryogenesis
Morphogenic competence
Caulogenic or rhizogenic Embryogenic: IPEDC, PEDC, EDC, NEC
Plant growth regulators
Buds: cytokinins / auxins Roots: cytokinins / auxins
IPEDC strong auxin PEDCPEDC weak auxin EDC
Initial cell Multiple cells, cell cluster Single cell or a few cellsDevelopment process stages
Induction – caulogenic initials meristemic center primordium
bud
Induction – EDC proembryo globular scutellar
coleoptile
Morphological polarity
Monopolarshoot apex or root apex
Bipolar plumule – radicle
Provascular bundle
Open type Closed type (half closed)
Subpathways Direct, indirect, intermediated D. In. Int. repetitive
Regenerant Adventitious buds, roots Embryoid, somatic embryo
Nursery Microcutting: rooting to plantlet SE-plantlet conversion to embling
There are hundreds of papers related to Banana tissue culture. It is unfeasible to review all the publications. Here, we are intend to introduce three major fields initially developed by Professor SuProfessor Su--ShienShien MaMa in National Taiwan in National Taiwan UniversityUniversity.
Spend the past sixty years in Plant Propagation and Plant Tissue Culture
Three major fields of Banana Tissue Culture developed by Professor MaⅠ. Somatic tissue and reproductive tissue derived organogenesis
1. Decapitated shoot apex culture (Ma and Shii, 1972; 1974)2. Inflorescence section culture (Ma et al., 1978; 1985)3. Vegetative and reproductive apex meristem culture (Ma et
al., 1978; 1980).
Ⅱ. Young male flowers derived somatic embryogenesis (Ma,1985; 1988; 1989).
Ⅲ. Programmed cell death and, rescuing culture and somaticembryogenesis in endosperm culture of diploid bananas.
Part IVegetative apex culture derived organogenesis
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Fig. 1. The induction of adventitious buds in shoot apex culture following decapitation and treated with cytokinin (Ma and Shii, 1972).
Regeneration medium
MS salts, organics
340 mg L-1 NaH2PO4
100 mg L-1 tyrosine
160 mg L-1 Adenine sulfate
2 mg L-1 of both IAA and kinetin
8 g L-1 agar
bud formation
Medium formBud No. / Sucker (shoot apex)
Intact Two divisions Four divisions
Sem-solid(0.8% agar)
9.5 11.7 20.9
Liquid medium(10 rpm rotator)
− 16.7 27.0
Table 2. Effect of explant division and medium form on adventitious bud number of banana AAA cv. Pei-Chiao
(Ma and Shii, 1974)
Fig. 2. Most of the buds transformed into corm-like growth on semi-solid medium (0.8%) due to water and nutrient stresses. (Ma and Shii, 1972.)
Some of adventitious bud were shooting and developed to plantlets, and survived in pot.
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Fig. 3. The corm-like buds induced to sprouting on reducedagar concentration medium (0.5%)
Fig. 4. Liquid medium and rotation stimulating bud sprouting and rooting of adventitious buds. A: agar medium, B-E: liquid medium, F: sole PGR liquid medium, G: Double distilled water
(Ma and Shii, 1974)
(cm
/ 3
wks
)
TreatmentTreatment
complete Salts plus sugar
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Fig. 5. Rapid shooting and rooting of adventitious budsin liquid medium (regardless bud age)
Days in culture
Ave
rage
hei
ght o
f pla
ntle
ts (c
m) 3 6
10 months
The development and application of in vitro organogenesis
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1. Healthy nursery production.2. Mass conal multiplication. (Huang et al., 1984)² Since 1983 conducted by TBRI 2-3 x 106 TC plants/yrs. In 1996, 20 x 106 TC plants/world)
3. In vitro selection of disease resistant variety Fusarium wilt race 4 resistant variety (TBRI, 1992).
4. Potential for in vitro breeding.5. In vitro germplasm conservation² FAO manual- minimal growth (Vuylsteke, 1989) ² INIBAP clonal repository- restricted growth (Belgium)
Part ⅡYoung male flowers derived somatic embryogenesis
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1. Four culture steps and defined medium of reproductive tissue culture.
2. Cyclic growth and embryogenic competence in suspension cells of banana AAA and AAB cultivars.
3. Acid growth model and Ma criteria of quality somatic embryo production.
Advantages and propagated character of male flower derived somatic embryogenesis1. Reproductive tissue display pluripotent in
vitro morphogenesis and high embryogenic competence.
2. Cell morphological transformation revealed high multiplication rate.
3. Mass clonal propagation of vigor uniform type somatic embryo.
4. The desirable mother stock can be identified before explant collection.
5. Unwant organ, without sacrifice the mother plant, and fruit production.
The culture procedures for male-flower derived somatic embryoenesisSteps Media1. Embryogenic callus induction
• Tip 20 male flower clusters.• 2 mm section
DedifferentiationCallus formation(MA1, M1, YC) semi-solid
2. Cell suspension culture• Orbital shaker 110 rpm conditional
medium (new/old 7:5) 2 wk interval subculture.
ProliferationMaintenance (MA2, M2, TB5) liquid
3. Somatic embryo formation• Plating• filter paper / 1/30 ml PCV per petri dish
or gelrite 0.3%
Regenerationembryo formation(MA3, A3, SH3)liquid 6-8 ml
4. Embling establishment • semi-solid, • liquid, • TIS
ConversionSE à plantletEmbling establishment(MA4, M4)
Table 3. The medium composition for the four culture steps in male flower derived somatic embryogenesis of bananas. (Ma, 1988; 1989)
Composition (mg/L)
(Symbols)
Callus induction(MA1, M1, YC)
Suspension proliferation(MA2, M2, TB5)
Embryoidformation(MA3, M3, SH3)
Emblingestablish(MA4, M4)
Salts and organics MS MS SH MS
Glutamine 100 100 100 100
d-biotin 1 1 1 1
Malt extract 100 100 100 100
Proline - - 230.2 -
2,4-D 2 1 - -
NAA 1 - - 0.5
IAA 1 - - -
2-iP - - 0.2 -
Kinetin - - 0.1 -
Zeatin - - 0.05 -
BA - - - 0.5
Lactose (g/L) - - 10 -
Sucrose (g/L) 30 45 45 30
Agar (g/L) 7 - - 7
Fig 4. The features in male flower derived granular callus formation (1), somatic embryo formation (2), SE-plantlet conversion (3), and embling establishment (4)(Ma, 1988).
1 2
3 4
Table 3. The classification of growth phases and characteristics in cell suspension population of bananas (Musa AAA and AAB cultivars.)
Growth phases Growth types* Stages Morphological characteristics Size (μm)
Proliferation(apolar or polar greowht)
Type Ⅰ 1 Nonembryogenic free cell 30×30 ~ 150×40
Type Ⅱ 2 Embryogenic free cell 20×20 ~ 30×20
Type Ⅲ 3 Bicellular
60×504 Multicellular
5 Cell cluster cell mass, PEM
Globularization Type Ⅳ
6 Globular budies 70×60 ~ 125×100
7 Oblong flatten 130×100 ~ 200×100210×100 ~ 300×200
8 Flatten elongated 300×150 ~ 570×230
Pseudoembryo
9 Initial dark elongated 450×350 ~ 560×200
10 Dark elongated 420×260 ~ 500×350
11 Dark pear-Shoped Ⅰ 450×350 ~ 800×450
12 Dark pear-Shoped Ⅱ 0.9×0.8~ 1.8×1.4mm
13 Dark pear-Shoped Ⅲ >1.8×1.4mm
Releasing(multiplication) TypeⅤ
14 Disorganization budding fragmentation
15 Releasing free cells, cell clusters
Cited from Huang et al. 1996; Huang et al 1999.*Growth types designated by Georget et al.(2000) corresponded to the Growth phase
Free cell, bicellularMulti-cellular Cell cluster
Cell massglobular Oblong
bodiesFlattenbodies
Flattenelongated
PrimaryDark elongated
Dark elongated
DarkPrar-Shaped ⅠDark
Pear-shaped Ⅱ
DarkPear-shaped Ⅲ
Nonembryogeniccells
Free cell cluster, mass
Disorganization transformant
Disorganization mass
Disorganization body
releasing body
Polar growth cyclePathway ⅠB
Pathway Ⅱ
Pathway Ⅲ
Apolar growth cyclePathway I A
Fig. 5. The cyclic developmental processes in various cell lines of bananasAAA and AAB genomic groups.
Fig. 6. The cyclic growth modeⅠ,Ⅱ,Ⅲ in embryogenic suspension cell cultures of banana AAA cv. Robusta and AAB cv. Fen-Chiao
Cycle 1 Cycle 2
Cycle 3
(Huang 1994)
AAB ‘Fen-Chiao’
Mesh Cultivar No of SE dish per ml PCV Size (mm) Singuleor
cluster Color Conversion(%)
30 ~ 60
AAB ‘Fen-Chiao’ 18,090 0.85 × 0.6 S + White 80
AAA ‘Pei-Chiao’ 18,000 0.55 × 0.45 + 2/3 White 50
AAA ‘Robusta’ 20,400 0.40 × 0.3 +++, +++ 1/2 White 10
< 60
Fen-Chiao (AAB) 40,710 0.75 × 0.5 S White 90
Pei-Chiao (AAA) 16,380 0.65 × 0.5 S White 70
Robusta 27,600 0.65 × 0.5 S, + White 80
> 30
Fen-Chiao 4,260 0.75 × 0.45 S, + 2/3 White 30
Pei-Chiao 39,000 0.30 × 0.35 +++ 1/3 White 10
Robusta 69,600 0.25 × 0.2 ++++ 1/5 White 5
Table 4. The effect of suspension cell cluster size on the developmentof somatic embryogenesis.
S: singular, +: 3 ~ 5, ++: 5 ~ 10, +++: 10 ~ 20, ++++: > 20 SE
Fig. 7. Auto-regulated hemostasis in extracellular pH level specific to growth phases during subculture duration of embryogenic cell suspension cultur of AAA ‘Pei-Chiao’ and AAB ‘Raja’. A1, B1: globular. A2, B2: proliferation. A3: cell releasing (Chung et al., 2006)
3. Acid growth model and Ma criteria of quality somatic embryo production
G, pH 4.8 – 5.3
P, pH 4.0 – 4.8
R, pH 3.3 – 4.0
Fig. 8. The extracellular pH change curve in the cell lines of banana AAA and AAB cultivars during a long-term maintenance culture.
(Chung et al., 2006)
Proliferation phase
Proliferation phase
Cell releasing phase
Cell releasing
Growth phases
Globularization
Figure 9. Cyclic development pathway and auto-regulated acid growth model inembryogenic cell suspension culture of triploid bananas.
H+
H+
H+
Proliferation
The strong proton efflux (pH<4.6) is assumed to interfere the establishment of physiological biopolarity and conducting to apolar proliferation. The proton influx or higher extracellular pH(>4.6) is the precondition for initiation of polar axis and destining to proembryogenesis. (The solid arrow represent trends of proton influx and developmental phase change, the dash arrow represent the strength of proton efflux and cyclic phase changes).
5.45.25.04.84.64.44.24.03.83.63.43.23.0
(Chung et al., 2006)
Fig. 10. The growth phase inter-conversion mediated extracellular pH manipulation during the cell suspension culture
(Chung, 2001)
Pre-treatment at pH 3.3
Pre-treat. at pH 4.9
(21 d)
Fig. 11. Cell releasing and polar growth of embryogenic cells by the fluorescent expressions of cell’s callose
(Chung, 2001)
Proliferation or Globulization Releasing
Cell releasing
Polar growth
Table 5. The distribution and attribute of initil cell division on the free cells of cell releasing phase were directed to pH 4.95 ± 0.2.
Percentage
I st. division 2 nd. division Equal division Unequal division T- shape (three cells)
CK (pH 3.30) 62.5 (168/269) 33.5 (90/269) 4.0 (11/269)
pH 4.95 2 days 10.6 (53/499) 38.9 (149/499) 50.5 (252/499)
4 days 4.5 (37/831) 27.1 (225/831) 68.4 (569/831)
Cell length (µm) apical 28.1± 6.8 31.0 ± 5.7 25.6 ± 2.4
basal 27.4 ± 6.2 22.7 ± 4.8 19.4 ± 3.5
Axis ratio (large / small) 1.07 ± 0.07 1.31 ± 0.16 1.32 ± 0.12
The size ratio of two daughter cell equals to or larger than 1.2 was categorized as unequal division.
(Chung, 2001)
Fig. 12. The effects of acidic pre-treatment duration on somatic embryo formation of banana AAB ‘Raja’. (Lu, 2001)
0
5,000
10,000
15,000
20,000
25,000
30,000
35,000
40,000
0 7 14 21 28 35 42 49
Pre-treated days
Num
ber o
f som
atic e
mbr
yos
(0.0
33 m
l PCV
per
dish
)
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Fig. 13. The influence of pre-treated pH levels and duration in different growth phases on somatic embryo formation of banana AAB ‘Raja’.
Releasing phase(pre-treated at pH 5.0)
Proliferation phase(auto-regulated pH)
Globulization phase(pre-treated at pH 3.3)
Quality criteria of somatic embryo
∗ Singular ∗ Synchronous (uniform)∗ Nomal (morphological bipolarity)∗ Stable (genetic)∗ High conversion rate (SE-plantlets)∗ Universal (various cultivars)
-- health, vigor
High quality somatic embryo produce system
Embryonic cell suspension culture
Plating
GerminationPlantlets
Pre-treat.polar growth
Globulartion phase
Quantitative SE
1 x 106 SE / 1 ml PCV
Quality SE
High conversion rate
Vigorembling
The potential impact in single cell origin of somatic embryogenesis
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1. High multiplication rate of cell morphological transformation (CMT)
EDC morphological transformation
Somatic embryo Emblingsomatic embryogenesis
1mL CPV 106 somatic embryos
2. Development of artificial synthetic, clonal asexual triploid seeds
A few synthetic triploid seed factory enough to
world plantation nurseryprovide
3. Improvement via in vitro breedingØEasy manipulation of in vitro mutagenesisØEfficient for in vitro selection of desirable solid mutant benefit to improve novel cultivar of triploid banana cultigens.
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4. In vitro molecular breeding Ø CMT is the basis of cell genetic transformation (CGT)
Ø Single cell origin of CGT CMT
solid transgenic plants.
5. Cryopreservation of banana germplasmØ PEDCs or EDCs, somatic embryosØ Cryopreservation / clonal repository in a limited space.
6. BiotransformationØ PEDC biosynthesis of useful bioproducts.