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.