Climate Change and Future Food Security: The Impacts on root and Tuber Crops
In Vitro Conservation and Cryopreservation in Tuber Crops · 2019-11-18 · • India holds a rich...
Transcript of In Vitro Conservation and Cryopreservation in Tuber Crops · 2019-11-18 · • India holds a rich...
In Vitro Conservation and
Cryopreservation in Tuber Crops
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Root and tuber crops are second only in importance to cereals as a global source of carbohydrates.
They also provide some minerals and essential vitamins.
The variability of various root and tuber crops is decreasing due to
• drastic changes in food habits
• cropping pattern, deforestation
• excessive land utilization
• extinction of landraces due to the overwhelming popularization of improved varieties
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Root and Tuber Crops Production
France Portugal
Morocco China
Thailand
Malaysia
Japan
Indonesia Papua New Guinea
Ethopia
Congo
Nambia Bolivia
Peru
Columbia
Mexico
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
There are five major areas of distribution of root and tuber crops in India.
(i) South-western hilly and coastal region
(ii) Southern peninsular region
(iii) Eastern coastal region
(iv) North-eastern region
(v) North-western region
Maximum species diversity is noticed in North-eastern and South-western hilly and coastal region.
• India holds a rich genetic diversity of tropical root and tuber crops, namely, cassava, sweet potato, aroids, yams and several minor tubers.
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Botanical name Common name Family Regions of distribution
Manihot esculenta Crantz
Cassava Euphorbiaceae Southern, occasionally in North-eastern regions
Ipomoea batatas (L.) Lam.
Sweet potato Convolvulaceae Introduced, found all over the country
Dioscorea alata L. Greater yam Dioscoreaceae Southern and North-eastern regions
Dioscorea rotundata Poir.
White yam Dioscoreaceae Introduced, Southern and North-eastern regions
Dioscorea esculenta (Lour.) Burk.
Lesser yam Dioscoreaceae Southern, North-eastern and eastern regions
Dioscorea bulbifera L. var. sativa
Potato yam Dioscoreaceae Southern, North-eastern and eastern regions
Colocasia esculenta (L.) Schott.
Taro Araceae Throughout India with greater diversity in North-eastern, eastern and southern region
Xanthosoma sagittifolium (L.) Schott.
Tannia Araceae Southern and eastern India
Important root and tuber crops in India
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Botanical name Common name Family Regions of distribution
Amorphophallus paeoniifolius (Dennst.) Nicolson
Elephant foot yam
Araceae Southern, North-eastern and eastern regions
Coleus parviflorus Benth. Chinese potato Labiatae Southern regions
Pachyrhizus erosus (L.)Urban
Yam bean Fabaceae North-eastern region
Moghania vestita Benth. ex Bak.
Moghania Fabaceae North-eastern region
Psophocarpus tetragonolobus (L.) DC.
Winged bean Fabaceae Southern and North-eastern regions
Maranta arundinacea L. Arrowroot Marantaceae Introduced, adapted to plains with high rainfall
Canna edulis Ker-Gawler Queensland or purple arrowroot
Cannaceae Introduced, adapted to high rainfall regions
Alocasia macrorrhiza (L.) G. Don
Giant taro Araceae Southern and North-eastern regions
Important root and tuber crops in India
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Conservation of Tuber Crops Genetic Resource
(National Status)
Crop Accessions (Nos.)
Cassava 1211
Sweet Potato 1124
Yams 1110
Edible Aroids 683
Minor Tuber crops 200
ICAR-CTCRI, Kerala
Crop Accessions (Nos.)
Cassava 601
Sweet Potato 1927
Taro 733
Yams 453
Edible Aroids 430
Minor Tuber crops 438
AICRP on Tuber Crops
(Source: Annual report 2017-18)
(Source: Annual report 2015-16)
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Conservation of Potato Genetic Resource
(National Status)
Crop Accessions (Nos.)
a) Tuberosum (Cultivars / parental lines)
Cultivars bred at CPRI 60
Advanced hybrids 96
Indigenous varieties 107
Indigenous samples 97
Exotic 2840
b) Andigena 762
c) Wild/semi-cultivated spp.
540 (125 species)
(Source: CPRI Annual report 2018-19)
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
At ICAR-CPRI, Shimla, 4502 accessions of cultivated and wild potato species, obtained from 30 countries, are conserved
Conservation of Tuber Crops Genetic Resource
(ICAR-NBPGR Status)
In field genebank, 283 accessions are being maintained at RS, Thrissur
In in vitro genebank, 518 accessions (~7000 cultures) are being maintained
Crop spp. Species Accession (No.)
Cultures (No.)
Sweet Potato (Ipomoea batatas)
1 261 3000
Dioscorea spp. 10 153 1850
Taro (Colocasia esculenta)
1 90 1000
Xanthosoma sagittifolium
1 10 100
Alocasia indica 1 4 50
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Nodal explants are excised, disinfected
and cultured
Check for
Contamination
Bacteria-free Clones
Cultures are established in 4-12 weeks, depending on genotype,
often requiring repeated disinfection
Disinfect
Discard
In vitro Culture Establishment in Sweet Potato
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Protocols developed/ refined for in vitro multiplication/ regeneration
In vitro multiplication/ Regeneration
Ipomoea batatas Dioscorea
alata
D. bulbifera
D. nipponica
D. esculenta
Colocasia esculenta
Alocasia macrorrhiza
Xanthosoma sagittifolium
D. wallichii
D. floribunda
D. pentaphylla
D. rotundata
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Protocols developed for In vitro
Conservation of Tuber Crops
Protocols developed for In vitro
Conservation of Tuber Crops
Normal Growth
D alata
D bulbifera
D pentaphylla
D rotundata
Colocasia esculenta
Slow Growth
Ipomoea batatas
D floribunda
Storage Organs Induction
Colocassia esculenta
Dioscorea spp.
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Protocols developed for In vitro
Conservation of Tuber Crops
Plant species Strategy Response Reference
D. alata MS + 0.15mg/l NAA at 25° C 10-12 months Mandal et al., 2000
D. bulbifera MS + 0.15mg/l NAA at 25° C 10-12 months Mandal et al., 2000
D. pentaphylla MS + 0.15mg/l NAA at 25° C 10-12 months Mandal et al., 2000
D. rotundata MS + 0.5 mg/l BAP + 0.1 mg/l NAA at 25° C
10-12 months Mandal et al., 2000
Colocasia esculenta
MS + 0.5 mg/l BAP + 0.1 mg/l NAA at 25°C
6-12 months Hussain et al., 2007a
Normal Growth
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Protocols developed for In vitro
Conservation of Tuber Crops
Plant species Strategy Response Reference
D. floribunda MS + 0.5 mg/l NAA at 18° C 10-12 months Mandal et al., 2000
Ipomoea batatas
MS + 0.2mg/l Kn + 0.1mg/l IAA + 4% sucrose (at 18° C)
16-18 months Mandal and Chandel, 1991
I. batatas MS + 0.2mg/l Kn + 0.1mg/l IAA + 4% sucrose (at 25° C)
6-12 months Mandal and Chandel, 1991
I. batatas MS + 0.2mg/l Kn + 0.1mg/l IAA + 2.0-2.5% sucrose (at 18° C)
6-12 months Mandal et al., 2000
I. batatas MS + 1.5- 2.0 % Mannitol at 25° C 6-12 months Mandal et al., 2000
Slow Growth
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
• A shoot tip, embedded deep into the corm tissue encircled with leaf primordial, and basal part of the petioles (size: 5-10 mm diam, 20-30 mm length)
Explant
• M1 = MS (3% sucrose) + 2.2 μM BAP + 0.6 μM NAA + 0.8% agar
• M2 = MS (8% sucrose) + 2.2 μM BAP + 0.6 μM NAA + 0.8% agar
• M3 = MS (10% sucrose) + 2.2 μM BAP + 0.6 μM NAA + 0.8% agar
Media
• Morphological Markers: 12 qualitative morphological traits such as corm weight, corm bud colour, fresh weight of plant, corm length (cm), corm weight (g) no. of eyes/corm
• Molecular markers: RAPD and ISSR markers
Genetic Stability
In vitro corm induction
Regrown on MS+ 1.5 mg/l BAP, 0.2 mg/l NAA and 0.2 mg/l GA3
Thawing at 40°C for 2 min
Plunge in LN
Air dehydration in laminar flow for 5½ hrs
Encapsulation in 3% Calcium alginate & osmoprotection in liquid MS medium containing
0.75 M sucrose for 1-4 days
Pre-culture on MS medium having 0.3M sucrose
Shoot tips (1-2 mm)
4-6 week-old in vitro mother cultures
Encapsulation
dehydration
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Protocols developed for
Cryopreservation of Tuber Crops
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Regeneration of encapsulated shoot tips of D. bulbifera after cryopreservation
Sucrose Concentration (M)
PRECULTURE DURATION (Days)
1 2 3
0.50 8.9 ± 5.7 cd 14.4 ± 8.3 c 17.8 ± 7.9 bc
0.75 21.1 ± 7.4 b 43.3 ± 8.2 a 45.6 ± 8.3 a
1.0 4.4 ± 4.9 de 0.0 e 0.0 e
Dehydration Duration (hrs.)
PRECULTURE DURATION (Days)
1 2 3 4 7
4 14.4 ± 8.3 35.6 ± 9.6 38.9 ± 7.4 37.8 ± 6.3 30.0 ± 6.7
5 20.0 ± 9.4 43.3 ± 8.2 46.7 ± 9.4 52.2 ± 9.2 40.0 ± 9.4
5½ 22.2 ± 9.2 45.6 ± 8.3 48.9 ± 9.9 56.7 ± 8.2 41.1 ±10.9
6 20.0 ± 9.4 37.8 ± 7.9 40.0 ± 9.4 47.8 ± 9.2 36.7 ±13.3
Effect of Preculture/ Dehydration Duration
Effect of Sucrose Concentration Mukherjee et al. (2009) Cryoletters
Plant species
Culture
system
Response
Reference
%survival % regeneration
Dioscorea alata Shoot tips 71 21 Mandal et al., 1996
D. rotundata Shoot tips 88 67 Mandal et al., 1999
D. floribunda Shoot tips 75 29 Mandal, 2000;
Mandal and Ahuja-
Ghosh, 2007
D. wallichii Shoot tips 64 37 Mandal et al., 1996
Ipomoea
batatas
Somatic
embryos /
embryogenic
tissue
62% Regeneration of plants from
cryopreserved embryogenic tissue
Mandal et al., 2000
Protocols developed for
Cryopreservation of Tuber Crops
Encapsulation dehydration Encapsulation dehydration
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Regrowth
Unloading solution
Thawing at 40°C for 2 min
Plunge in LN
PVS2 for 0-130 min
Treat with loading solution (LS)
Pre-culture on MS medium having high sucrose
Shoot tips (1-2 mm)
4-6 week-old in vitro mother cultures
Vitrification
Figure: Regrowth of Nodal explants of Dioscorea rotundata after cryopreservation using Vitrification technique; a) Control, b) Vitrification control, c) Cryopreserved Nodal Explants showing regrowth, d) fully grown plantlet from cryopreserved nodal explant
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
Effect of exposure time to PVS2 at 0°C on survival and regeneration of shoot tips of D. bulbifera
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
PVS2 Exposure Time (min)
-LN + LN
Survival (% ± SD)
Regeneration (% ± SD)
Survival (% ± SD)
Regeneration (% ± SD)
0 95.6 ± 4.9 a 81.1 ± 5.7 a 0.0 d 0.0 d
50 92.2 ± 6.3 ab 72.2 ± 6.3 b 63.3 ± 8.2 c 27.8 ± 7.9 c
70 91.1 ± 7.4 ab 65.6 ± 6.8 c 78.9 ± 7.4 b 44.4 ± 8.3 b
90 90.0 ± 6.7 b 64.4 ± 4.9 c 88.9 ± 8.7 a 57.8 ± 6.3 a
110 85.6 ± 8.3 c 58.9 ± 5.7 d 81.1± 9.9 b 45.6 ± 8.3 b
130 81.1 ± 7.4 d 50.0 ± 9.4 e 74.4 ±10.6 b 28.9 ± 10.9 c
Mukherjee et al. (2009) Cryoletters
Plant species
PVS2 Treatment
Duration (min)
Response Reference
% survival % regeneration
Dioscorea alata 90 min 82 39 Mukherjee et al.,
2009
D. bulbifera 90 min 89 58 Mukherjee et al.,
2009
D. rotundata 110 min 89 71 Mandal et al.,
2008
D. wallichii 90 min 71 28 Mandal et al.
2000
Protocols developed for
Cryopreservation of Tuber Crops
(Vitrification Technique) (Vitrification Technique)
Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR Eighth International Training on In vitro and Cryopreservation Approaches for Conservation of PGR
TARO: FROM LAB TO LAND
Kerala farmers’ request to Dr. Joseph K. John regarding
land races of taro
PRIMARY HARDENING AT NBPGR
ESTABLISHMENT OF PLANTLETS AT RS- THRISSUR
TARO IN MEDIUM TERM CONSERVATION
AT IN VITRO GENE BANK
AT FARMER’S FIELD
Supplied 40 accessions of Colocasia esculenta
(including landraces)
ICAR-NBPGR Regional Station - Thrissur, Kerala
for “On Farm Conservation”
Genetic diversity assessment D. alata
170 150
500
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
M 18 19 20 21 22 23 24 25 26 27 28 29
260 230 200
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
M 18 19 20 21 22 23 24 25 26 27 28 29
In vitro conserved D. alata accessions were analysed for genetic
diversity using genomic and EST-SSR markers.
Dioscorea alata accessions IC-
044217 and IC-044214; IC-
044235A and IC-044233
showed maximum similarity of
98% .
Genetic Diversity Analysis of Sweet Potato
Coefficient
0.60 0.70 0.80 0.90 1.00
EC-5589 EC-324082
EC-64037 EC-13468 EC-19716 EC-55037
EC-17159 EC-4717 IC-582674
EC-13969-A EC-13469-A EC-105211 EC-339350
EC-669408 EC-30928-P30 EC-324078 EC-30928-P9
EC-394614 IC-427623 EC-4160-P11 EC-105212
EC-394610 EC-55040 EC-27277 IC-582670
EC-13969 EC-30927-P8 EC-31600
EC-394606 EC-394600 IC-582676 EC-339353
EC-394613 EC-339352
• Genetic diversity analysis in in vitro conserved I batatas accessions was done using EST-SSR markers
• The accessions viz. EC-324082 (CIP, New Delhi), EC-64037 (NBPGR, Regional Station, Amravati), EC-13468 (NBPGR, Regional Station, Amravati) were found to be identical
Acknowledgements Dr. B B Mandal
Dr. Neelam Sharma
Dr. Zakir Hussain
Dr. Vartika Srivastava
Mr. Devendra K Nerwal
Mr. Suresh Chand Mali