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


Root and Tuber Crops Production

France Portugal

Morocco China

Thailand

Malaysia

Japan

Indonesia Papua New Guinea

Ethopia

Congo

Nambia Bolivia

Peru

Columbia

Mexico


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.


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


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


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


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)


Conservation of Potato Genetic Resource

(National Status)


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)


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


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


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


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.




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




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



• 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



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


Protocols developed for

Cryopreservation of Tuber Crops


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



Encapsulation dehydration Encapsulation dehydration


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


Effect of exposure time to PVS2 at 0°C on survival and regeneration of shoot tips of D. bulbifera


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



(Vitrification Technique) (Vitrification Technique)


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

in vitro conservation and cryopreservation in tuber crops · 2019-11-18 · • india holds a rich...

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