Does Cryopreservation Stress Impact Genotype Integrity? A Case Study with Germplasm of Musa spp - Third International Symposium on Plant Cryopreservation, Asia Hotel, Bangkok, Thailand March 26-28, 2018

1. Anuradha Agrawal, R. Sanayaima, R. Goswami, J. Goenka, D.P. Meena, Shivani, R.
Singh, S. Uma1 and Rishi Kumar Tyagi2
National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi – 110 012, INDIA
1National Research Centre for Banana (ICAR-NRCB), Tiruchirapalli, Tamil Nadu, INDIA
2 Coordinator, Asia-Pacific Consortium on Agricultural Biotechnology and Bioresources
(APCoAB), Asia-Pacific Association of Agricultural Research Institutions (APAARI)
Bangkok 10100, THAILAND
Does Cryopreservation Stress Impact Genotype Integrity?
A Case Study with Germplasm of Musa spp.
ThirdInternationalSymposiumonPlantCryopreservation
AsiaHotel,Bangkok,Thailand,March26-28,2018

2. TripuraTamil Nadu
Kerala
Assam
Musa spp. (bananas and plantains)
An important food and fruit crop in India

3. • Immense economic importance and diversity (>300 cultivars,
several endemic wild species like M. cheesmannii, M. sikkimensis,
M. nagensium and M. andamanica)
• Loss of habitat and local landraces
• Climate change and other unknown biotic and abiotic stresses
Urgency for Musa Conservation in India
Musa arunachalensis: a new species of section
Rhodochlamys from Arunachal Pradesh, India
Ref: Sreejith et al. Phytotaxa 134 (1): 49–54 (2013)
Arunachal Pradesh, India
wild bananas

4. IndianCouncilofAgriculturalResearch(ICAR)Network
National Bureau of
Plant Genetic
Resources (NBPGR)
National Research Centre on
Banana (NRCB)

5. Musa Conservation in Complimentary Mode
Field Gene Bank of NRCB, Trichy, India
(1993)
Cryobank of NBPGR, New Delhi,
India (2004)
•Absence of seeds in most of the triploid
Musa cultivars
•Predominantly vegetatively propogated
In Vitro Genebank of NBPGR, New
Delhi, India (1986)

6. Comparison of cryopreservation protocols in banana
cv Robusta (AAA)
Ref: Agrawal A., Panis B., Swennen R. (2004) CryoLetters 25: 101-110
Simple
freezing of
PM
Vitrification
of PM
Droplet
Vitrification
of PM
Droplet
Vitrification
of IM
0
20
40
60
80
100
120
C LN C LN C LN C LN
SF V DV DV-IM
Shootregeneration(%)
% Callus
% Shoot
a
d
b
bc
bc
bc
b
b

7. Proliferating
‘cauliflower’ like
meristematic clump
Individual
meristem
HighBAPorTDZ
Rootedplantsoncharcoal
Ease in isolation Some genotypes
recalcitrant
Requires great skill
for isolation
Droplet Vitrification
of Proliferating
Meristems
Droplet Vitrification
of Individual
Meristems
Good recovery
rate, dependent on
good Isolation

8. Studies on regenerated plants from cryopreserved
meristems of Musa
• Survival
• Regrowth of shoot
• Recovery of plantlets
• Hardening and field transfer
• Morphological evaluation of regenerants
• SSR analysis of regenerants

9. Framework of DST Project (2004-2009)
0
2
4
6
8
10
12
14
16
Matti(AA)
VaiBalhaKaul
(AA)
Kodappanilla
Kunnan(AB)
Somai(AB)
Sommrani
Monthan(ABB)
PidiMonthan
(ABB)
EnnaBenian
(ABB)
KalluMonthan
(ABB)
Barjahaji(AAA)
Karpura
Chakkarakeli
(AAB)
No.ofshoots
0
1
2
3
4
5
6
7
8
9
10
Chenkadali
(AAA)
H-2(AAB)
BoothiBale
(ABB)
Giant(AAB)
AbharVelchi
(AAB)
Nendran-I
(AAB)
Nendran-II
(AAB)
Nendran-III
(AAB)
Nendran-IV
(AAB)
Nendran-V
(AAB)
No.ofshoots
0
2
4
6
8
10
12
14
16
Agniswar(AB)
BhatManohar
(ABBB)
Soniyal(AAB)
Adukkan(AB)
Manoranjithana
(AAA)
SannaChenkadali
(AA)
Musaaurantiaca
Nendran-VII(AAB)
Nendran-VIII(AAB)
Kodappanillakunnan
(AB)
No.ofshoots
Accessions with green
bars yielded proliferating
meristems
Shoot multiplication rate on MS + BAP (100 µM) medium
(First Batch of 20 accessions)

10. 0
20
40
60
80
100
120
C LN C LN C LN C LN C LN
30 min. 60 min. 90 min. 120 min. 150 min.
Survival(%)
PVS2
Callus
Shoota
e
a
de
a
cd
b
cd
bc
e
Pidi Monthan (ABB)
0
20
40
60
80
100
120
C LN C LN C LN C LN C LN
30 min. 60 min. 90 min. 120 min. 150 min.
PVS2
Survival(%)
Callus
Shoot
cd
a
a a a
cd
cd bc
ab
d
Sommrani Monthan (ABB)
0
20
40
60
80
100
120
C LN C LN C LN C LN C LN
30 min. 60 min. 90 min. 120 min. 150 min.
PVS2
Survival(%)
Callus
Shoot
a
e
ab
de
a
cd
a
cd
bc
c
Kallu Monthan (ABB)
Agrawal A. et al. (2008) Current Science 94:1125-1128
Vitrification Protocol in Indian Cooking Bananas (Monthan subgroup)

11. Descriptors Recorded (IPGRI 1996)
1 Leaf habit,
2 Pseudostem color
3 Pseudostem appearance
4
Predominant underlying color of the
pseudostem
5
Pigmentation of the underlying
pseudostem
6 Wax on the leaf sheaths
7 Appearance of the leaf upper surface
8 Color of leaf upper surface
9 Color of leaf lower surface
10 Wax on leaves
11 Insertion point of leaf blades on petiole
12 Shape of leaf blade base
13 Color of midrib dorsal surface
14 Peduncle color
15 Peduncle hairiness
Contd.
16 Male bud type
17 Male bud shape
18 Male bud size
19 Bract
20 Bract apex shape
21 Bract imbricate
22 Color of bract external face
23 Color of the bract internal face
24 Color on the bract apex
25 Male flower behavior
26 Compound tepal basic color
27 Compound tepal pigmentation
28 Lobe color of compound tepal
29 Lobe development of compound
tepal
30 Free tepal color
31 Free tepal shape
FlowerVegetative

12. 32 Free tepal appearance
33 Free tepal apex appearance
34 Free tepal apex shape
35 Anther exertion
36 Filament color
37 Style basic color
38 Pigmentation on style
39 Style exertion
40 Style shape
41 Stigma color
42 Ovary shape
43 Ovary basic color
44 Ovary pigmentation
45 Dormant color of the male
flower
46 Bunch position
47 Bunch shape
48 Bunch appearance
49 Rachis type
50 Rachis position
51 Fruit shape
52 Transverse section of fruit
53 Fruit apex
54 Remains of flower relics at
fruit apex
55 Pedicel surface
56 Fusion of pedicels
57 Immature fruit peel color
58 Mature fruit peel color
59 Adherence of the fruit peel
60 Cracks in fruit peel
61 Pulp color before maturity
62 Pulp color at maturity
63 Fruit fall from hands
64 Flesh texture
Flower Fruit

13. Cryopreserved banana cv Pidi Monthan (ABB) with fruiting bunch, at
NBPGR, New Delhi (left) and NRCB, Trichy (right)

14. NRC on Banana, TrichyNBPGR, New Delhi
Cryopreserved plants with fruiting bunch, at NBPGR, New Delhi (left) and NRCB,
Trichy (right)
Musa ABB cv ‘Sommrani Monthan’

15. Parameters
TC
Control
Cryo
Control
LN
treated
Sucker
Control
MSE
C.D @
1%
CV %
Pseudostem Height 275.00 281.75 282.00 282.50 8.45 6.68 1.04
Pseudostem Girth 62.00 61.50 60.75 62.00 5.45 NS 3.79
No. of leaves at Shooting 13.75 15.00 15.50 14.75 0.33 1.32 3.89
Petiole length 62.75 63.00 63.75 61.25 1.12 2.43 1.69
Leaf area in sq. m 14.65 15.97 16.93 15.98 345.32 1.35 3.70
Time taken for shooting 362.25 366.50 363.50 366.00 67.90 NS 2.26
Time taken for bunch
maturation
115.00 115.00 113.75 113.00 22.78 NS 4.18
Duration 477.25 481.50 477.25 479.00 42.00 NS 1.35
Bunch weight (in kg) 15.62 16.25 15.62 15.75 0.26 NS 3.23
No. of hands/ bunch 8.75 8.50 8.50 8.75 0.36 NS 6.96
No. of fruits / hand 13.25 13.75 13.75 13.25 0.28 NS 3.92
Total no. of fruits 116.00 116.75 116.75 116.00 81.25 NS 7.75
Growth and Yield Data of Cryopreserved Musa accession (ABB)
Agrawal A. et al. (2011) Acta Horticulturae 908: 129-138

16. Sommrani Monthan (ABB)
Fruit from Non-
Cryopreserved
Plant (Ash
coated fruits)
Fruit from
Cryopreserved
Plant
(Green fruits)
Agrawal A. et al. (2011) Acta Horticulturae 908: 129-138

17. What caused the reversion?
Stress from tissue culture or cryopreservation
(PVS2, Freeze stress, Thawing injury)?

18. Sl. No. Primer name Primer sequence
1. AGMI 33 AGTTTCACCGATTGGTTCAT
AGMI 34 TAACAAGGACTAATCATGGGT
2. AGMI 35 TGACCCACGAGAAAAGAAGC
AGMI 36 CTCCTCCATAGCCTGACTGC
3. AGMI 67 ATACCTTCTCCCGTTCTTCTTC
AGMI 68 TGGAAACCCAATCATTGATC
4. AGMI 93 AACAACTAGGATGGTAATGTGTGGAA
AGMI 94 GATCTGAGGATGGTTCTGTTGGAGTG
5. AGMI 95 ACTTATTCCCCCGCACTCAA
AGMI 96 ACTCTCGCCCATCTTCATCC
6. AGMI 103 ACAGAATCGCTAACCCTAATCCTCA
AGMI 104 CCCTTTGCGTGCCCCTAA
7. AGMI 129 GGAGGCCCAACATAGGAAGAGGAAT
AGMI 130 CATAAACGACAGTAGAAATAGCAAC
8. Mb SSR 1-146 F CCGTTGGATTTCTCCCCCACA
Mb SSR 1-146 R GAAGAACTGGGCTTACCCAGGA
9. Mb SSR 1-149 F CCGAAACGAAGGTTACAACAA
SSR primers used for Sommrani Monthan analysis

19. SSR analysis for the cryopreserved Sommrani
Monthan (ABB)
Lanes 1, 6, 11, 16, 21, 26, 31, 36 & 41 : SMTC
Lanes 2, 7, 12, 17, 22, 27, 32, 37 & 42 : SMCC
Lanes 3, 8, 13, 18, 23, 28, 33, 38 & 43 : SMCM
Lanes 4, 9, 14, 19, 24, 29, 34, 39 & 44 : SMFS
Lanes 5, 10, 15, 20, 25, 30, 35, 40 & 45 : Green Mutant
Lanes 1 – 5 : AGMI 33/34
Lanes 6 – 10 : AGMI 35/36
Lanes 11 – 15 : AGMI 67/68
Lanes 16 – 20 : AGMI 93/94
Lanes 21 – 25 : AGMI 95/96
Lanes 26 – 30 : AGMI 103/104
Lanes 31 – 35 : AGMI 129/130
Lanes 36 – 40 : Mb SSR 1-146 F/ R
Lanes 41 – 45 : Mb SSR 1-149 F / R

20. Green mutant
Ash colour fruit
•Our initial study showed
that 100% of the in vitro-
conserved and 93.3% of the
cryopreserved Musa
germplasm was genetically
stable.
•But the single plant
reversion prompted further
studies to be undertaken in
other accessions.

21. Cryopreservation of Sucker Meristems (SM)
by Droplet Freezing Technique in Musa ABB cv. Karpura Chakkarakeli

22. 0
20
40
60
80
100
120
C LN C LN C LN
SF VIT DF
Survival(%)
Callus
Shoot
b
ba ab
a
a
0
20
40
60
80
100
120
C LN C LN C LN
PM IVM SM
Survival(%)
Callus
Shoot
a
a
a
a
a
a
2 months8 months14 months
Use of sucker meristems
(SM) for cryopreservation
of banana was successful
The duration of
cryopreserving plants from
SM was only 2 months and
this cuts down the cost of in
vitro maintenance of
cultures
Amongst the three
cryopreservation
methods, droplet
vitrification method
gave the highest
number of shoot
during regeneration
Agrawal et al., 2014. In Vitro Cellular and Developmental Biology – Plant 50:345–356

23. Tissue culture control Fruiting of cryopreserved plant
derived from proliferating meristems
Fruiting of cryopreserved plant derived
from in vitro single shoot meristems
Fruiting of cryopreserved plant
derived from sucker meristems
Fruiting of cryopreserved plants in Karpura Chakkrakeli (ABB)

24. Primer Sequence
Melting
temperatur
eTM (ºC)
Annealing
temperature
TA (ºC)
No. of
scorable
bands
BN-01F AAG AAG GCA CGA GGG TAG 56
BN-01R CGA ACC AAG TGA AAT AGC G 56
BN-02F GGA AAA CGC GAA TGT GTG 54
57.4 1
BN-02R AGC CAT ATA CCG AGC ACT TG 60
BN-03F CGT CAC AGA AGA AAG CAC TTG 62
50.9 2
BN-03R CCT CTC CAT CGT CAT CAA TC 60
BN-04F GAG CCC ATT AAG CTG AAC A 54
BN-04R CCG ACA GTC AAC ATA CAA TAC A 62
BN-05F TGA GGC GGG GAA TCG GTA 58
BN-05R GGC GGG AGA CAG ATG GAG TT 64
BN-06F AAG AAG GCA CGA GGG TAG 56
60 2
BN-06R CGA ACC AAG TGA AAT AGC G 56
BN-07F GGA AAA CGC GAA TGT GTG 54
50 1
BN-07R AGC CAT ATA CCG AGC ACT TG 60
BN-08F ATG TCG CTT CGG ACC AGA 56
58.5 3
BN-08R GCA GGA CGA AGA ACT TAC C 58
BN-09F ATG ATC ATG AGA GGA ATA TCT 56
50.3
1
BN-09R TCG CTC TAA TCG GAT TAT CTC 60
BN-10F GGT TGG AAC GGA GGT ATA CTA A 64 50.9 2
BN-10R TCC AAG CTT ATC GAT CTA CG 58
Genetic stability
Analysis using SSR
Primers

25. Primer Sequence TM (ºC) TA (ºC)
No. of scorable
bands
BN-11F TGT CGA AGC ATC CTA CAT C 56
56 1
BN-11R CTT GGA AAC ATG AGA AAC ATA C 60
BN-12F TTG AAG TGA ATC CCA AGT TTG 58
50.9 2
BN-12R AAA ACA CAT GTC CCC ATC TC 58
BN-13F TGC TCT TCC ACA TCT CAA GAA C 64
59.8 1
BN-13R GAT TGC ACG GAG ATT CAA CA 58
BN-14F GGT GCT CTT CGG AGG A 52
59.8
BN-14R CGC TTT ATA TCC ATT CCC A 54
BN-15F GAG CCC ATT AAG CTG AAC A 56
59 1
BN-15R CCG ACA GTC AAC ATA CAA TAC A 62
BN-16F GAT GAT GGT GAG AGG CTG ATG A 66
60
BN-16R GGT CGG TAT GGG AAG CAC C 62
BN-17F TTT GCC TGG TTG GGC TGA 56
50 3
BN-17R CCC CCC TTT CCT CTT TTG C 60
BN-18F GAG CCC ATT AAG CTG AAC A 54
60 1
BN-18R CCG ACA GTC AAC ATA CAA TAC A 62
BN-19F CTC CTT TGT GAG CTC GGC ATA T 66
50.3 1
BN-19R AGG GTC CAA GAA ACT CCT CCA A 66
BN-20F GAA GCA TCC AAT GGA CCT A 56
60 1
BN-20R GCG AAC TCA CAA TAG CGA 54
SSRprimers
used

26. Primer Sequence TM (ºC) TA (ºC)
No. of scorable
bands
BN-21F CCC GTC CCA TTT CTC A 50
59.8 1
BN-21R TTC GTT GTT CAT GGAATC A 52
BN-22F GGT GGA TGG CTG GGT A 52
59.8 1
BN-22R GGA TCC AAG CTT ATC GAG TT 58
BN-23F GGT GCT CTT CGG AGG A 52
58.5 1
BN-23R CGT TTA TAT CCA TTC CCA 50
BN-24F GCT TGT CTC TCA CCC ACT C 60
50 1
BN-24R ACC GAC TCC CCAATA GG 54
BN-25F CTG GTC CTT TTC AGT TCA CTC 62
50.3 3
BN-25R TAG GCA GCT CCC AAT CA 52
BN-26F CTA GGC TTC CTG CTG CTC 58
60
BN-26R TGA GCG AAT TTG ATC AGAAC 56
BN-27F GCA CGAAGA GGC ATC AC 54
54.3 2
BN-27R GGC CAAATT TGA TGG ACT 68
SSRprimers
used

27. SSR Profile of Cryopreserved Plants
BNR 13
BNR 17
BNR 18
In all the
cryopreserved plants
regardless of the
method used, plants
show 92% similarity,
based on 24 SSR
primer pairs

28. 0
10
20
30
40
50
60
70
80
90
100
Sommrani
Monthan
(ABB)
Pidi Monthan
(ABB)
Kallu
Monthan
(ABB)
Booditha
Bontha (ABB)
Karpura
Chakkarakeli
(ABB)
H-2 (AAB) Barjahaji
(AAA)
Kodappanilla
Kunnan (AB)
Vai Balha Kaul
(AA)
C
LN
Genetic stability study using SSR applied to diverse accessions (9) of
Musa, cryopreserved using Droplet Vitrification of PM

29. Primer Sequence
Melting temperatureTM
(ºC)
Annealing
temperature
TA (ºC)
Ma-1-132 GGA AAA CGC GAA TGT GTG 54
57.4
AGC CAT ATA CCG AGC ACT TG 60
Ma-SSR-18 CGT CAC AGA AGA AAG CAC TTG 62
50.9
CCT CTC CAT CGT CAT CAA TC 60
Ma-SSR-07 AAG AAG GCA CGA GGG TAG 56
60
CGA ACC AAG TGA AAT AGC G 56
Ma-SSR-07 GGA AAA CGC GAA TGT GTG 54
50
AGC CAT ATA CCG AGC ACT TG 60
Ma-1-136 ATG TCG CTT CGG ACC AGA 56
58.5
GCA GGA CGA AGA ACT TAC C 58
Ma-1-127 ATG ATC ATG AGA GGA ATA TCT 56
50.3
TCG CTC TAA TCG GAT TAT CTC 60
Ma-1-5 GGT TGG AAC GGA GGT ATA CTA A 64 50.9
TCC AAG CTT ATC GAT CTA CG 58
SSR primers used (21)
Contd…

30. Primer Sequence TM (ºC) TA (ºC)
Ma-1-230 TGT CGA AGC ATC CTA CAT C 56 56
CTT GGA AAC ATG AGA AAC ATA C 60
Ma-1-27 TTG AAG TGA ATC CCA AGT TTG 58 50.9
AAA ACA CAT GTC CCC ATC TC 58
Ma-1-18 TGC TCT TCC ACA TCT CAA GAA C 64 59.8
GAT TGC ACG GAG ATT CAA CA 58
Ma-2-2 GAG CCC ATT AAG CTG AAC A 56 59
CCG ACA GTC AAC ATA CAA TAC A 62
Ma-1-16 TTT GCC TGG TTG GGC TGA 56 50
CCC CCC TTT CCT CTT TTG C 60
Ma-1-24 GAG CCC ATT AAG CTG AAC A 54 60
CCG ACA GTC AAC ATA CAA TAC A 62
Ma-2-4 CTC CTT TGT GAG CTC GGC ATA T 66 50.3
AGG GTC CAA GAA ACT CCT CCA A 66
Ma-3-41 GAA GCA TCC AAT GGA CCT A 56 60
GCG AAC TCA CAA TAG CGA 54
Contd…

31. Primer Sequence TM (ºC) TA (ºC)
Ma-3-41 CCC GTC CCA TTT CTC A 50 59.8
TTC GTT GTT CAT GGA ATC A 52
Ma-3-50 GGT GGA TGG CTG GGT A 52 59.8
GGA TCC AAG CTT ATC GAG TT 58
Ma-3-55 GGT GCT CTT CGG AGG A 52 58.5
CGT TTA TAT CCA TTC CCA 50
Ma-3-59 GCT TGT CTC TCA CCC ACT C 60 50
ACC GAC TCC CCA ATA GG 54
Ma-3-79 CTG GTC CTT TTC AGT TCA CTC 62 50.3
TAG GCA GCT CCC AAT CA 52
Ma-3-90 GCA CGA AGA GGC ATC AC 54 54.3
GGC CAA ATT TGA TGG ACT 68

32. L M1 M2 M3 M4 M5 M6
a b c d a b c d a b c d a b c d a b c d a b c d
L M7 M8 M9
a b c d a b c d a b c d
a=control, b-d=cryopreserved samples
290 bp
SSR profile obtained with primer pair Ma-3-41

33. 0
10
20
30
40
50
60
70
80
90
100
Sommrani
Monthan
(ABB)
Pidi Monthan
(ABB)
Kallu
Monthan
(ABB)
Booditha
Bontha (ABB)
Karpura
Chakkarakeli
(ABB)
H-2 (AAB) Barjahaji
(AAA)
Kodappanilla
Kunnan (AB)
Vai Balha
Kaul (AA)
Graphical representation of similarity coeffiecient based in UPGMA
analysis of the cryopreserved banana samples of 9 accessions, as
compared to the control (non-cryopreserved samples)

34. Conserving Banana Diversity
for Use in Perpetuity
Bioversity International Funded Project (2009-12)
Agrawal A. and R.K. Tyagi. 2014. Int. J. Innovative Horti. 3(2):115-133.

35. ABB and
AAB
groups
0
10
20
30
40
50
60
70
80
Pelipita
BlueTorresStraitIsland
Cachaco
Fougamou1
Kayinja
PisangRadjah
KelongMekintu
Akpakpak
ObubitNtangagreen
mutant
PlantainNo.2
MaidenPlantain
Meanshootregeneration(%)
a
abc
bc
bc
d
ab
ab
a
cd
cd
d
0
10
20
30
40
50
60
70
80
90
Kasaka
PisangMulik
PisangMadu
PisangMadu
PisangTongat
TjauLagada
Pu-teLa-Bum
BieYeng
PisangBuntal
GuNinChio
Pitu
luaiLepMuNang
Sowmuk
Meanshootregeneration(%)
a a
ab ab ab ab
ab ab
bc
cd
cd
AA
group bc
Accessions selected
for stability studies

36. Treatments (4) (3 samples per treatment)
SSR Primers (11)
Ma-SSR-18, Ma-SSR-07, Ma-1-127, Ma-1-18, Ma-2-2, Ma-1-24, Ma-3-41,
Ma-3-55, Ma-3-59, Ma-3-79, Ma-3-90
Plants grown T1 Tissue Culture Controls (not subjected to any
cryotreatment)
T2 PVS2 treated plants (not subjected to LN
treatment)
T3 Plants obtained after LN treatment using
vitrification technique
T4 Plants obtained after LN treatment using droplet-
vitrification
No. of plants : 3 per treatment
Design RBD
Planting Date : July, 2011
Flowering and
Fruiting
Nov., 2012 – Feb, 2014
(First and ratoon crop)

37. T1: In Vitro
conserved
T2 : Cryopreserved using vitrification
T4 :Cryopreserved using droplet- vitrification
Pelipita(ABB)
ANOVA revealed that most of the traits of frozen material were non-significantly
different from non-frozen controls and in vitro conserved material
T3: PVS2 treated
Field Characterization

38. TC-C1 TC-C2 TC-C3 PVS-C1 PVS-C2 PVSC3 V-LN1 V-LN3 V-LN3 DV-LN1 DV-LN2 DV-LN3
SSR profile obtained with primer pair Ma-1-18
1000 bp
500 bp
200 bp
200 bp
EC653573,
Pisang Madu
EC653545,
Pelipita
EC653551,
Pisang
Radjah

39. EC653545, Pelipita (ABB)
Similarity coefficient based
in UPGMA analysis >80%

40. EC653551, Pisang Radjah (AAB)
Similarity coefficient based
in UPGMA analysis >80%

41. EC653573, Pisang Madu (AA)
Similarity coefficient based in UPGMA
analysis >80%, except in one sample
of TCC (64%)

42. •20 amplified fragments detected (average 1.8 per primer) among all
control and cryopreserved samples.
•UPGMA (Nei and Li/Dice) analysis at the SSR loci exhibited a high
(>85%) co-efficient of similarity among the control and cryopreserved
samples in the 3 accessions tested.
•In all the three accessions, one tissue culture control sample
exhibited lower co-efficient of similarity (64-85%) as compared to the
rest of the samples.
•All the bands were found to be monomorphic, and variation
observed was due to the absence of the band in a few samples; no
variation was observed based on amplicon size.
•The SSR data is being compared with the morphological data to
conclude if variation observed was in the coding or non-coding region
of the genome.

43. Musa AABp cv Nendran
Cryobank
the
bananas…
But keep a
watch!
Conclusion

44. Genomic
Group
Indigenous accessions (46) Exotic accessions (30)
AAA Borjahaji, Chenkadali,
Dwarf Cavendish, Manoranjitham
Chinese Cavendish, Cocos
Grand Naine, Palang, Pisang Nangka,
Biu Ketip, Red Dacca, Robusta
AAAp and
AAAh
Nendran Akpakpak, Maiden Plantain, Mbi
Egome 1, Obubit Ntanga green
mutant, Plantain no.2, Guineo
AAB Abhar Velchi, Alpan, Amritpani,
Dasaman, Digjowa, Dudhsagar, Giant, H-
2, Honda, Kalibow, Kottavazhai, Ladan,
Ladan Pointed, Soniyal
Blue Torres Strait Island, Cocos,
Kelong Mekintu, Pisang Keling,
Pisang Radjah
ABB Boothi Bale, Booditha Bontha Batheesa,
Cherapunji,
Enna Benian, Karpura Chakkarakeli, Kallu
Monthan, Pacha Bontha Batheesa, Pidi
Monthan, Sommrani Monthan, Sakkar
Chinia, Thozhouvan, Udhyam,
Vellapalayankodan
Cachaco, Kayinja, Pelipita, Fougamou
1
Musa germplasm cryobanked (using meristems) at NBPGR
(As up to 2017)
Contd…

45. Genomic
Group
Indigenous
accessions
Exotic accessions
AB Adukkan, Agniswar,,
Kodappanilla Kunnan,
Somai
AA Matti, Sanna
Chenkadali, Vai Balha
Kaul
Bebek, Bie Yeng, Gu Nin Chio, Gulum,
Kasaska, Kluai Lep Mu Nang, Lidi, NBA 14,
Pisang Buntal, Pisang Mulik, Pisang Tongat,
Pisang Madu, Pitu, Pu-te La-Bum, Sowmuk,
Tjau Lagada
AAw Musa aurantiaca M. acuminata type 2 x, M. ac. ssp
malaccensis type malaccensis; M. ac. type
Kluai Thong Det, M. ac. type Pisang Cici Alas,
M. ac. ssp. banksii
BBw M. balbisiana, Butuhan intermediate apex
Australimusa M. basjoo, M. textilis, M. jackeyi

46. Acknowledgements
• Organizers, CryoSymp2018
• Director General, ICAR, Director(s), NBPGR and
NRCB
• Bart Panis, K.U. Leuven, Belgium
• Bioversity International, Rome, Montpellier,
Leuven & Delhi
(Emile Frison, Stephan Weise, Nicolas Roux,
Prem Mathur, Anne Vezina , Ines van den
Houwe)
• Department of Science and Technology
• S. Uma, R. Selvarajan, M.S. Saraswathi (NRCB),
India
• Anuradha Agrawal and staff/students of TCCU,
NBPGR, India
Thank you!
Projects funded by :
Indian Council of
Agricultural Research
Department of Science and
Technology, GoI
Department of
Biotechnology, GoI
World Bank , NATP
Bioversity International
Global Crop Diversity Trust