Credit Seminar (Hort 591) Date: 26-10-16

Fruit crop improvement through tissue culture techniques- Achievements and Future

prospects

Kuldeep PandeyL-2015-A-85-M

Department of Fruit SciencePunjab Agricultural University, Ludhiana

Plant Tissue Culture

Plant tissue culture refers to the cultivation of plant organs, tissue or cells in vessels using artificial media

Also referred to as cell, in vitro, axenic or sterile culture

The theoretical basis for plant tissue culture was proposed by Gottlieb Haberlandt in his address to the German Academy of Science in 1902

Shoot bud regeneration from Tobacco suspension culture was reported by White in 1939

First plant from the mature plant cell was regenerated by Braun in 1959

Most of the modern tissue culture media have been derived from the work of Skoog and coworkers during 1950s and 1960s

History of Plant Tissue Culture

The first embryo culture was done by Hanning in 1904 who cultured mature embryo of some crucifers

This technique was soon applied by Laibach in 1925 recover hybrid progeny from interspecific cross of in Linum

Haploid plants from pollen grains were first produced by Maheswari and Ghua in 1964 by culturing anthers of Datura

In 1960, Cocking produced large quantities of protoplast by using cell wall degrading enzyme

Application of Tissue culture techniques in Fruit crop improvement

Recovery of virus free plant

Somatic embryogenesis

Somaclonal variation

Embryo culture

Anther culture

Somatic hybridization

Cryopreservation

Micro-grafting

Recovery of virus free plant

Viruses are eliminated by:

A.Thermotherapy

B.Cryotherapy

C.Chemotherapy

Species Virus Therapy ReferenceP. dulcis cv. Labanese

Candidatusphytoplasma

In vitro thermotherapy Chalak et al(2005)

P. armeniaca PPV, PDV, PNRSV

In vitro thermotherapy Laimer et al(2006)

P. ameniaca cv. Bebecou

PPV In vivo and in vitro thermotherapyshoot tip culture

Koubouris et al(2007)

P. avium PNRSV, PDV In vivo thermotherapy Howell et al(2001)

P. avium cv. Early River

ACLSV, PNRSV, PDV

In vitro thermo, chemotherapy

Cies´lin´ska (2007)

P. domesica cv. CacanskaLepotica

PPV In vivo chemotherapy Paunovic et al(2007)

P. domesica cv. Earliblue

PNRSV In vitro thermotherapy Dziedzic (2008)

Plant species Virus eliminatedAnnanas sativus (Pineapple) Unspecified Fragaria sp (Strawberry) Complex of viruses, crinkle, edge, vein

banding, yellow virus complex, pallidosis

Malus sp (Apple) Latent viruses , Apple chlorotic leaf spot virus

Musa sp (Banana) Cauliflower mosaic virus, Bunchy top virus, Musa mosaic virus

Rubus ideaus (Raspberry) Mosaic

Vitis vinifera (Grape) Grapevine fan leaf virus, Arabis mosaic virus

Important Fruit Crops in which virus elimination has been successful using meristem culture

(Khurana et al 2003)

Somatic Embryogenesis

Somatic embryogenesis has four well recognized phases:

(i)Induction(ii)Development(iii)Maturation(iv)Germination

Somatic embryogenesis and plant regeneration from immature embryos of Carica papaya x Carica cauliflora cultured in vitro

Fig- Morphology and isozymes of Carica papaya, C. cauliflora, and interspecific hybrids of these two species derived from embryo culture: (A) Hybrid plant(B) C. papaya plant(C) C. cauliflora plant(D) Leaves of (left to right) C. papaya, hybrid, and C. cauliflora.

(Chen et al 1999)

Somatic embryogenesis and plant regeneration inPsidium guajava L. cv. Banarasi local

.

(Rai et al 2007)

(A) Zygotic embryo explant from 10-week-old fruit (B) Globular to cotyledonary stage somatic embryos (C) Maturation of somatic embryos (D) Initiation of germination of somatic embryos (E) Plantlets obtained by germination of somatic embryos (F) A healthy and well-hardened plant of guava

Species Explant ReferenceP. mume cv. Nanko Immature

cotyledonTsukamoto et al(2007)

P. avium Zygotic andSomatic embryo

Garin et al(1997)

P. avium Zygotic embryo Talleux et al(1999)

P. cerasus Immaturecotyledon

Tang et al (2000)

P. avium Protoplast David et al (2006)

P. persica cv. Bell of Georgia, Bailey, Tennessee Natural, Nemaguard, Encore

Zygoticembryo

Srinivasan andScorza (2007)

P. incisa cv. February Pink Leaf, root Cheong andPooler (2004)

P. subhirtella autumn rosa Petiole Machado et al(1995)

Cultivar(s) Explant Response PGRs Media Reference

Cambodiana, Carabao, M20222,Turpentine N2-1-4-3, Earlibush,Chino, Turpentine N2-1-7-2,Sabre and Ono

Ovules Somatic embryogeneis(SE) and precociousgermination

With or Without1–2 mg/l ; 20%coconut water

Modified MS medium+400 mg l−1 glutamine+100 mg l−1 ascorbic acid

Litz et al(1982)

Amrapali Nucellus SE and germination

5–20 μM BAP MS and B5 media

Laxmi et al(1999)

Carabao and Manila Super

Nucellus SE Modified Medium

Patena et al(2002)

Ambalavi Nucellus SE and plantletregeneration

0.25 mg/l BAP+ 1.0 mg/l NAA;0.5 mg/l 2ip

Modified medium (BM)

Chaturvediet al (2004)

Important fruit crops in which protocols are being standardized for efficient cloning of plants through

somatic embryogensisMango (Mangifera indica)Banana (Musa sp.)Citrus (Citrus sp.) Grapes (Vitis venifera) Pineapple(Annanas sp.)Papaya (Carica papaya)PomegranateLitchi (Litchi chinensis)Apple(Malus domestica)Peach(Pyrus sp)Strawberry(Fragaria sp.)Datepalm (Phonix dectylifera)Walnut (Jaglans regia)Ber (Ziziphus sp.) (Singh 2011)

Somaclonal variation

Somaclonal variants may arise due to:

1.Chromosomal structural changes2.Gene mutation3.Plasma gene mutation4.Gene amplification5.Karyotype changes6.Mitotic crossing over etc.

Application of Somaclonal variation1.Novel variants – Thornless Blackberry released in New Zealand as cv Lincoln Logan Hall et al (1986)

2.Disease resistance- Banana- F. oxysporium sp.cubens Apple- Phytopthora cactorum Peach- Xanthomonas comp.pv.pruni Pseudomonas syringae pv syringae3.Abiotic stress resistance a)Cold tolerance b)Salt tolerance c)Drought tolerance d) Aluminum tolerance

4.Herbicide resistance5.Insect resistance6.Seed quality

Cavendish banana cultivars resistant to Fusarium wilt acquired through Somaclonal variation in Taiwan

(Hwang et al 2004)

Name of the somaclonal variant

Cultivar from which derived

Usefulness Reference

CIEN-BTA-03 Cavendish Resistant to yellow Sigatoka

Trujillo and Garcia 1996

TC1-229 Tai-Chiao No. 1 Semi-dwarf and resistant to Fusarium wilt

Tang et al 2000

Tai-Chiao No. 1

Formosana

Giant Cavendish

Giant Cavendish

Resistant to Fusarium wilt

Resistant to Fusarium oxysporum f. sp. cubense (Foc)race 4; bunch 40% heavier

Hwang 2002

Hwang 2002

TC2-425 Tai-Chiao No. 2 Larger bunch size Hwang 2002

GC-138 Pei-Chiao Growing cycle 1–1.5 month shorter

Hwang 2002

------ Grand Naine Dwarf with superior quality

Stover 1987

Somaclonal Variation: a Useful Tool to Improve Disease Resistance of Pear Rootstock ‘Old Home × Farmingdale’ (OHF

333) (Pyrus communis L.)

Nacheva et al (2014)

Class 0 – plants without visual symptoms of the infection;

Class 1 – plants in which the leaf cut has blackened;

Class 2 – plants in which the cut and the central nerve have blackened

Class 3 – plants in which the leaf and the leaf petiole have blackened

Class 4 – plants in which the apical shoot and the upper part of the plant have blackened

Class 5 – the whole plant is dying

Somaclone Degree of susceptibility

Somaclone Degree of susceptibility

Control 5 X-16 0

VIII-1 0 X-8 1

X-1 0 VI-2-1 1

VI-2 0 VI-3-1 0

VI-3 4 X-1-3 0

X-1-1 0 VIII-1-1 4

VI-1 0 X-1-4 0

VI-8 1 X-1-5 2

VIII-5 0 VI-1-1 4

VI-12 4 VI-3-2 2

Studies on Somaclonal Variation in Strawberry (Fragaria x ananassa Duch.) Cultivars

(Kaushal et al 2004)

Embryo CultureApplications of embryo culture:

1.Recovery of Distant Hybrids

2. Recovery of Haploids plants from interspecific crosses

3. Propagation of orchid

4. Shortening the breeding cycle

5. Overcoming dormancy

Some fruit Plants in where embryo culture used- Apple, Cherry, Pear, Peach, Grapes, Banana, Papaya (Singh 2005)

The first peach embryo culture studies (Davidson 1933, 1934)

i.Shortening the breeding cycle of peachii.Increasing germination of embryos from early ripening cultivarsiii.From interspecific crosses

Stage of fruit Media Reference

Immature fruitlets (35–45 days old)

MS basal medium(9 mg/l BA + 3 mg/l kinetin + 400 mg/l glutamine + 500 mg/l activated charcoal + 60 g/l sucrose

(Nathhang 1999). Chandra et al (2003)

Fruitlets at 6–8 weeks after pollination

MS medium(casein hydrolysate(1.25 g/l) and sucrose (4.5%).

Sahijram et al (2005)

Fig- Fruits of:(A)Seeded berries from C41-7(B)Parthenocarpic berries from C41-7(C)Mature berries from C41-5(D)Overripe berries from C41-5.

A sternosprmocapic seed less Vitis vinifera x Vitis routundifolia Hybrid Developed by

Embryo Rescue

Ramming et al 2000

Fig- Effect of maturity status and medium composition on callus induction in zygotic embryos of Pisang Jajee

Embryo rescue and plant regeneration in banana (Musa spp.)

(Uma et al 2010)

Fig-Effect of embryo maturity and media composition on plantlet regeneration from callus tissue derived from zygotic embryos

Anther/Pollen Culture

Application of anther culture:

1.Obtaining haploids2.Obtaining homozygous diploids

Regeneration of androgenic embryos in apple (Malus x domestica Borkh.) via anther and microspore culture.

(Hofer 2005)

CitrusMalus domestica Pyrus communisPyrus pyrifoliaPrunus persica Prunus aviumPrunus domestica Prunus armeniaca Vitis vinifera

Actinidia deliciosaOlea europaeaMorus alba Musa balbisiana (BB)Carica papaya Annona squamosa FeijoasellowianaOpuntia ficus-indicaEriobotrya japonica

Doubled haploid production in fruit crops

(Germana 2006)

Androgenic potential in coconut (Cocos nucifera L.)

(Perera et al 2008)

Fig- (a)Isolated rachilla (r) containing male (mf) and female flowers (ff)

(b) Close view rachilla

(c) Isolated male flower: Anthers are totally covered by the petals(p)

(d)Anthers (an) attached to the filaments (f)

Fig-(a) Embryos (em) derived from anther (an); (b) Mature embryo with germination point (gp); (c) Germinating embryo with a shoot (s) emerging by splitting of haustorium (h); (d) Calli (ca) bearing anther; (e) An anther bearing both callus (ca) and embryos (em); (f) Somatic embryos (se) developed from anther derived callus; (g) A complete plantlet derived from anther

Somatic Hybridization

The technique of somatic hybridization involves the following four steps:(i)Isolation of protoplast(ii)Fusion of protoplast(iii)Selection of hybrid cells(iv)Regeneration of hybrid plants

Applications of somatic hybridization

1.Symmetric hybrid2.Cybrid

(Cybrids) regenerated via symmetric protoplast fusion in Citrus

Fusion combination References

C. sinensis cv. Bonanza navel + C. sinensis cv. Valencia

Deng et al (2000)

C. sinensis cv. Newhall navel + C. sinensis cv. Anliu Deng et al (2000)

C. reticulata cv. Red tangerine + Yuzu (C. junos) Deng et al (2000)

Page tangelo + C. sinensis cv. Valencia Deng et al (2000)

Page tangelo + C. sinensis cv. Gailiang Deng et al (2000)

Swinglea glutinosaa + Sour orange (C. aurantium) Moreira et al (2000)

C. sinensis cv. Rohde Red Valencia’ + C. reticulata cv. Dancy

Moreira et al (2000)

C. reticulata cv. Cleopatra + Sour orange (C. aurantium) Moreira et al (2000)

Microcitrus papuana + Sour orange (C. aurantium) Xu et al (2004)

Fusion combination References

C. reticulata.cv. Shekwasha + C.sinensis cv. Valencia Guo and Grosser (2005)

C. sinensis cv. Salustiana + C. aurantifolia cv. Mexican lime

Fuster et al (2005)

Page tangelo + Rough lemon (C. jambhiri) Guo et al (2006) C. reticulata cv. Chios mandarin + C-35 citrange (C.sinensis× P. trifoliata)

Guo et al (2006)

C. reticulata cv. Chios mandarin + C-35 citrange (C.sinensis× P. trifoliata)

Dambier et al (2011)

C. reticulata cv. Chios + 4475 citrumelo (C.paradisi× P. trifoliata)

Dambier et al (2011)

C. reticulata cv. Chios + citrandarin (C. reticulata × P. trifoliata)

Dambier et al (2011)

C.aurantifolia cv. Macrophylla + Carrizo citrange (C.sinensis × P.trifoliata)

Dambier et al (2011)

Fusion combination References

Tommy Atkins+ Kensington Pride Rezazadeh et al (2011)

Keitt + Kensington Pride Rezazadeh et al (2011)

Haden + Kensington Pride Rezazadeh et al (2011)

wild pear + Colt cherry Ochatt et al (1989)

Rubus ideaus + R. fruticosus Mezzetti et al (2001)

Diospyrus glandulosa +D. kaki(persimmon)

Tamura et al (2002)

Synthetic Seed

The concept of synthetic seed was given by Murashige (1977), but first report on the development of synthetic seeds was published by Kitto and Janick (1982).

There are two types of synthetic seeds: HydratedDesiccated

Scope of Synthetic Seeds

Plant species Propagules encapsulated References

Actinidia deliciosa (Kiwifruit)

Apical and axillary buds Adriani et al (2000), Romay Alvarez et al (2002)

Ananas comosus (Pine apple)

Axillary buds Soneji et al (2002), Gangopadhyay et al (2005)

Carica papaya (Papaya) Somatic embryos Castillo et al (1998)

Citrus sp. Somatic embryos Antonietta et al (1999, 2007)

Malus pumila (Apple rootstock M.26)

Apical and axillary buds, Nodes, Shoot tips

Piccioni and Standardi (1995), Piccioni (1997), Capuano et al (1998),Sicurani et al (2001), Brischia et al (2002)

Mangifera indica (Mango) Somatic embryos Ara et al (1999)

Musa sp. (Banana) Shoot tips, Shoot apices, Somatic embryos

Ganapathi et al (1992), Rao et al (1993), Suprasanna et al (2001),Ganapathi et al (2001)

Plant species Propagules encapsulated

References

Psidium guajava (Guava)

Somatic embryos, Shoot tips, Nodal segments

Akhtar (1997), Rai and Jaiswal (2008), Rai et al (2008)

Punica granatum (Pomegranate)

Nodal segments Naik and Chand (2006)

Pyrus communis (Pear) Shoot tips Nower et al (2007)

Pistacia vera (Pistachio)

Somatic embryos and embryogenic mass

Onay et al. (1996)

Vitis vinifera (Grape) Somatic embryos Das et al (2006)

CryopreservationCryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection i.e., the selection through freezing of samples with special properties or cryotherapy i.e., the elimination of viruses from infected plants through apex cryopreservation.

Effect of cryopreservation on the sanitary state of a cv. Prunus rootstockexperimentally contaminated with Plum pox virus.

(Brison et al 1997)

Species Explant Reference

P. dulcis cv. Ferragnes, Supernova

Cell suspension Damiano et al (2003)

P. armeniaca Dormant bud Irina et al (2006)

P. avium Embryonic tissue March(2005)

P. avium In vitro shoot tip Shatnawi et al (2007)

P. avium Seed Chmielarz (2009)

Elimination of grapevine virus A (GVA) by cryopreservation of in

vitro-grown shoot tips of Vitis vinifera L

(Wang et al 2003)

Low temperature storage of mango (Mangifera indica L.) pollen

(Dutta et al 2013)

Micro-grafting

(Naz et al 2007)

In vitro studies on micrografting technique in two cultivars of Citrus to produce virus free plants

Micrografting and ex vitro grafting for somatic embryo rescue and plant recovery in avocado

(Persea americana)

(Raharjo et al 2005)

Micrografting of almond (Prunus dulcis Mill.) cultivars “Ferragnes” and “Ferraduel”

(Yıldırım et al 2010)

Method of micrografting

Success of micrografts (%)“Ferragnes” “Ferraduel”

Vertical slit 90.0 100.0

Wedge on the stump

60.0 70.0

Effects of micro grafting method on success of micrograft

Effects of scion size on the success of almond micrografting

Size of microscion (mm)

Success of micrografts (%)“Ferragnes” “Ferraduel”

4 93.0 100.0

815

100.093.0

100.0100..

Species Virus Therapy ReferenceP. dulcis cv. Marcona, Fournat,uono, Ferraduel

PNRSV, PDV In vitro micrografting

Rizqi et al(2001)

P. persica PRSV, PDV, CLSV

In vitro shoot tip grafting

Navarro et al(1982)

P. persicacv. Suncrest

PNRSV In vitro micrografting

Heuss-LaRosaet al (1995)

Future Prospect of Tissue culture in Fruit crop breeding

Future prospects of tissue culture in fruit crop breeding appears to galore

i. In present scenario, where the growers face the problem of shortage of disease free planting material, the tissue culture is a potential means to tackle this problem

ii. Inoculation of AMF or other beneficial microbes during weaning

iii. Expanding the existing genetic resource in fruit crops

iv. Conservation of wild species