characterization of artificial-polyploid plants on yams

A member of CGIAR consortium www.iita.org

Characterization of artificial-polyploid

plants on yams

Maiko Matsumoto MSc. candidateKagoshima University Japan

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Outline

• Background• Objective• Methods• Results• Next step



Problems: Difficulty to breed yam

・ Disturb hybridization breeding 　　　　→ Flowers scarcity → Different flowering phase

Artificial polyploidy is a potential method to overcome these problems

Background

・ Dioecism → Making the breeding cycle very long → The sex is unclear until flowering



Is polypoidy breeding an effective method?

Polyploidy influences the phenotype, such asshape of leaf ,stoma and yield

Water yam (D. alata) : Phenotypic diversity within the species

The diversity of chromosomes number 　　　　 (Abraham and Nair, 1991; Gamiette et al., 1999 ; Babil et al., 2010)

Higher ploidy levels　 (Babil et al., 2010; Lebot et al.,2009;Toyohara et al.,2002)

Background

Diploid (2x=40)

Triploid (3x=60)

Tetraploid (4x=80)

(Leaf)Round

(Stoma) Large and low

density

(Yield)Increase



Water yam : Induction of polyploidy by colchicine treatment (Babil et al., 2011)

Diploid TetraploidColchicine

Background



←Stomata of the diploid parent water yam (No.132) and its tetraploid variant.

Babil et al., 2011. Japanese Journal of Tropical Agriculture 54(4): 132-139

→ 　 Artificial polyploidy influences the phenotype of leaf and stoma

← Leaf shape of diploid parent water yam (No.132) and its tetraploid varient

Background



August 2013 ～： In vitro plants with potential of polyploid had been generated by colchicine treatment(IITA)

February 2014 ～ : Identification of ploidy level and sub-cultured(TUA)

Phase I: Midori Iino: Generation of polyploids yam (TUA)

Collaborative research

April. 2013 ～ March. 2016

White and yellow guinea yam :

Background

Obvious influence of polyploidy



To evaluate the expression of phenotypes (leaf, stoma and yield) of artificial-polyploid plants induced by colchicines treatment in D. rotundata and D. cayenensis.

August 2014 ～ : Characterization of polyploids yam - In vitro multiplication of polyploids plantlets 　　　　　　　 (IITA) - Acclimatization 　(IITA) - Agro-morphological characterization of artificial-polyploid white and yellow yams plants 　(IITA-KU) - Chlorophyll content and yield (IITA)

Phase II: Maiko Matsumoto (KU)

Objective



Plant materialsSpecies Accession No Condition of plant No of in vitro

plantlets

D. rotundata TDr 1793 Check (No treated) 10

D. rotundata TDr 1793Polyploid

(Treated with colchicine 0.1%)

10

D. rotundata TDr 2351 Check 10D. rotundata TDr 2351 Polyploid 10D. rotundata TDr 2720 Check 10D. rotundata TDr 2720 Polyploid 10

D. cayenensis TDc 2790 Check 10D. cayenensis TDc 2790 Polyploid 10

Total No of plants 80・ Plants of check were treated with distilled water. ・ Artificial-polyploid plants were induced by 0.1% colchicine treatment under tissue 　　　 culture 　 conditions

METHODOLOGY



1 monthAcclimatization

Transplant in pot in SH

Morphological investigation at early growth stage

2 months

At highest growth stage

At harvest stage

Subculture

Backup

METHODOLOGY

in vitro plantlets



Morphological investigation item at early growth stage ( January 2015) Investigation using yam descriptor:

Young stem colour Young stem colour spot at spine base

→ Leaf width/length (IITA)

→ Basal internode length (IITA)

→ Guide vine length (IITA)　→ Chlorophyll content (IITA)

→ Stoma width/length/density (IITA –KU)

→ Cuticule thickness measurement on fresh leaves (KU)

METHODOLOGY



METHODOLOGY

Leave length and width Internode length Guide vine length

Morphological investigation item at Early growth stage (Dec. 2014)



Morphological investigation item at highest growth stage (March 2015)

・ Investigation using yam descriptor ｓ・ Chlorophyll content・ Stoma width / length / density

Morphological investigation of tuber (May 2015)

・ Investigation using yam descriptor ｓ・ No of tuber・ Fresh weight of tuber

METHODOLOGY



METHODOLOGYDATA PROCESSING

1. Data collected

5 weeks after Transplant to SH

Accession treat No.plant No.leafLeaf

width (cm)

Leaf length (cm)

Shape (length/width

)Size

(Length x width)

TDr1793 1 1 1 3.8 4.7 1.2 17.9TDr1793 1 1 2 4.3 3.4 0.8 14.6TDr1793 1 1 3 3.6 4.0 1.1 14.4TDr1793 1 2 1 3.2 3.3 1.0 10.6

TDr1793 1 2 2 3.6 4.0 1.1 14.4

2. Data processedSAS- Software-IITA

Proc Glm for ANOVAdata a;input acc $ 1-7 treat plnt leaf LW LL LSHAPE LSIZE;

cards;

Proc sort; by acc;run;

proc glm;class acc treat plnt leaf;Model LW LL LSHAPE LSIZE = acc treat plnt leaf acc*treat;means treat acc*treat / duncan;run;

Source DF Type III SS

Mean Square

F Value Pr > F

Acc 3 25.2 8.4 10.27 <.0001

Treat 1 0.02 0.02 0.02 0.8763

Plnt 2 3.49 1.74 2.13 0.1271

Leaf 2 8.82 4.41 5.40 0.0070

Acc*Treat 3 0.61 0.20 0.25 0.8621



PRELIMINARY RESULTSLeaf Width (cm) = LW

p ＜ 0.05Source DF Type III SS Mean Square F Value Pr > F

Acc 3 25.20 8.40 10.27 <.0001

Treat 1 0.02 0.02 0.02 0.8763

Plnt 2 3.49 1.74 2.13 0.1271

Leaf 2 8.82 4.41 5.40 0.0070

Acc*Treat 3 0.61 0.20 0.25 0.8621

1= Check

2 = Polyploid



PRELIMINARY RESULTSLeaf length (cm) = LL

p ＜ 0.05Source DF Type III SS Mean Square F Value Pr > F

Acc 3 14.16 4.72 4.87 0.0043

Treat 1 8.13 8.13 8.39 0.0053

Acc*Treat 3 0.53 0.17 0.18 0.9067

ANOVA

1= Check

2 = Polyploid



Leaf length (cm)PRELIMINARY RESULTS

TDr1793 TDr2351 TDr2720 TDc27900.0

1.0

2.0

3.0

4.0

5.0

6.0

＊ : p ＜0.05

＊

Check

Polyploid



PRELIMINARY RESULTSLeaf Length / Leaf Width (Leaf shape)

Source DF Type III SS Mean Square F Value Pr > F

Acc 3 0.05 0.01 0.91 0.4407

Treat 1 0.37 0.37 17.61 <.0001

Acc*Treat 3 0.01 0.006 0.30 0.8287

p ＜0.05

1= Check

2 = Polyploid



PRELIMINARY RESULTSlength/width (Shape)

TDr1793 TDr2351 TDr2720 TDc27900.0

0.2

0.4

0.6

0.8

1.0

1.2

＊ : p ＜0.05

＊＊＊

Check

Polyploid



PRELIMINARY RESULTSlength/width (Shape)

Leave from check plant: length larger

than width (Long)

Leave from polyploidy plant:

length less or equal than width (Round)



PRELIMINARY RESULTSBasal internode length (BINOCM)


Acc 3 60.91 20.30 13.70 <.0001

Treat 1 27.18 27.18 18.34 0.0001

Acc*Treat 3 16.48 5.49 3.71 0.0202

p ＜0.05

1= Check

2 = Polyploid



PRELIMINARY RESULTSBasal internode length (cm)

TDr1793 TDr2351 TDr2720 TDc27900.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

＊ : p ＜0.05

＊

＊

Check

Polyploid



PRELIMINARY RESULTSGuide vine length (cm)


Acc 3 1533.54 511.18 2.52 0.1037

Treat 1 3066.64 3066.64 15.10 0.0019

Acc*Treat 3 168.73 56.24 0.28 0.8410

p ＜0.05

1= Check

2 = Polyploid



PRELIMINARY RESULTS

TDr1793 TDr2351 TDr2720 TDc27900.0

10.0

20.0

30.0

40.0

50.0

60.0

p ＜ 0.05

Check

Polyploid

Guide vine length (cm)

＊＊



Multiplication of the polyploid material for future activity

In vitro plantlets

Acclimatization

Transplant in pot and culture for VC

Vine propagation

Mini tubers

Backup

Survival rateShoot formation rate

NEXT STEP

High temperature / water stress treatments



Thank you so much・ Supervisors: 　　 Dr. Lopez - Montes Antonio　　 Dr. Gueye Badara　　 Dr. Kikuno Hidehiko・ Genetic Resource Center・ Yam barn

・ International Institute of Tropical Agriculture ・ Tokyo University of Agriculture・ Kagoshima University


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characterization of artificial-polyploid plants on yams