Micropropagation in fruit crops

Central Institute for Subtropical Horticulture, Lucknow, India

Rajesh Pati PhD,Maneesh Mishra and Ramesh Chandra

e‐mail: rajeshpati777@gmail.com 

Introduction

• Micropropagation is a proven means of producing millionsof identical plants under a controlled and aseptic condition,independent of seasonal constraints.

• It not only provides economy of time and space but alsogives greater output and allows further augmentation of elitedisease free propagules.

• India is homeland of many important fruit crops such asIndian gooseberry (Emblica officinalis Gaertn), bael (Aeglemarmelos Corr.), Guava (, Psidium guajava), jamun orblack plum (Syzygium cuminii L. Skeels.), Mango(Mangifera indica) and Papaya (Carica papaya).

• Most of these crops have medicinal value and are suitablefor growing under marginal situations.

• The commercial production of these crops is restricted dueto the shortage of desirable planting material.

• Micropropagation can play an important role in rapidlyincreasing new cultivars of these fruit crops.

Aegle marmelos Corr.

Introduction• Aegle marmelos Corr., belongs to family Rutaceae, is moreprized for its pharmacological virtues than its edible quality.

• Because of pharmacological importance, it’s become potentialcandidate for developing transgenics to enhance its medicinalproperties.

• Maximum mortality of micropropagated plants occur duringacclimatization phase because plantlets undergo rapid andextreme changes in physiological functioning, histologicaland biochemical changes.

• In order to investigate the actual reason of this limitation, testsamples were collected at different micropropagation stages(In vitro, and acclimation).

Findings• Rapid clonal micropropagation protocol of Aegle marmelos Corr.cv. CISH-B1 and CISH-B2 was achieved by nodal stem segmentof mature bearing tree.

• Three centimeter long shoots having one axillary bud excisedfrom 10-15th nodal region of shoots during September gave quickin vitro bud burst (5.33 days) when cultured on MS mediumsupplemented with BAP, 8.84 μM + IAA 5.7 μM.

• The maximum number of proliferated shoots (9.0/explant) wereobtained on same medium supplemented with BAP 8.84 μM +IAA 5.7 μM.

• The micro shoots were rooted (100 %) on ½ strength MS mediumsupplemented with IBA 49.0 + IAA 5.7 μM. .

• In vitro rooted plants were acclimatized on coconut huskcontaining ½ strength MS plant salt mixture and under shadenet house (50 % shade 70-80 % RH).

• The plants were established in the field after acclimatization.

• The micropropagated plants were tested for its genetic fidelityusing 13 RAPD, 3 ISSR and 2 DAMD primers.

• Profile obtained by all the three Single Primer AmplificationReaction (SPAR) technique from mother tree andmicropropagated plants revealed genetic uniformity ofmicropropagated plants with that of mother tree.

F

DCBA

E

G

Fig: Micropropagation of Aegle marmelos (bael). (A) In vitro shoot budinduction, (B) proliferation, (C) Rooting, (D) Acclimatization, (E)Acclimatized plants (F) 3 month old acclimatized plants in polyhouse (G)Plant growing in field

Genetic uniformity

Histological and biochemical changes

• Maximum mortality of micropropagated plants occur duringacclimatization phase, because plantlets undergo rapid andextreme changes in physiological functioning, histologicaland biochemical changes.

• In order to investigate the actual reason of this limitation, testsamples (Leaf, stem and root) were collected at differentstages of micropropagated plants (In vitro and acclimation).

• The biochemical result showed that micropropagated plantletsproduced significantly low total chlorophyll (0.042 mg/g freshweight), reducing sugar (3.227%), NR activity (1.353NO2/h/g fresh weight) and but higher protein (0.048 μg/g)during in vitro phase.

• The in vitro raised plants showed abnormal histological featureslike altered leaf mesophyll, absence of thick cuticle, sunkenstomata, poorly developed stem and root histology.

• Photoautrophic mode of nutrition during in vitro phase increasedthe survival rate during acclimatization compared tophotoheterotrophic mode of nutrition

• Photoautotrophism phenemoneon has substantial influence onthe physiology and development of in vitro regenerated Aeglemarmelos Corr. plantlets.

Anatomy of leaf

(A) Whole mount of in vitro leaf showed open type stomata with fully turgidguard cells, (B) whole mount of acclimatized leaf showed partiallyclosed stomata.

A B

(A) T.S. of in vitro leaf, showed single layered epidermis with almost no cuticle andsingle layered and poorly developed palisade parenchyma with poorlydeveloped vascular tissue.

(B) T.S. of acclimatized leaf, showed single layered epidermis with thick cuticle andwell developed double layered palisade mesophyll while the lower side hadspongy parenchyma with air spaces and sunken type stomata.

BA

Anatomy of leaf

Anatomy of stem

(A) T.S. of in vitro stem, had a polystelic structures, vascular bundles were arrangedin a ring and they were conjoint collateral and open no corck cambium, showedprimary medullary rays, uniseriate epidermis, no cuticle, distinct endoderm wasabsent while cortex was parenchymatous.

(B) T.S. of acclimatized stem, well developed cork cambium, parenchymatous pithbut the pith was mucilaginous, distinct secondary growth with well developedwood and large woody vessels, epidermis was uniseriate and covered with cuticle,and cortex was collenchymatous.

A B

Anatomy of root

(A) T.S. of in vitro root, showed poorly developed pith, undifferentiated cortex withvery less amount of storage tissue and no secondary growth at all.

(B) T.S. of acclimatized Root, had a well developed parenchymatous cortex with tannincells, pericycle is made of stone cells vascular cylinder consisted of secondaryxylem towards inner side and secondary phloem towards outer side, secondaryxylem showed wide vessels scattered among trachieds, medullary rays were presentwhile pith was negligible.

A B

Psidium guajava (L.)

Introduction• Guava (Psidium guajava Linn.) commonly known for its

food, nutraceutical and commercial values throughoutthe world.

• The guava plant parts are used for the development ofvarious industrial and pharmaceutical products.

• Guava contains broad spectrum of phytochemicalsincluding polysaccharides, vitamins, essential oils,minerals, enzymes, proteins, sesquiterpenoid alcoholsand triterpenoid acids , alkaloids, glycosides, steroids,flavanoids, tannins, saponins .

• Guava is very rich in antioxidants and vitamins and alsohigh in lutein, zeaxanthine and lycopene .

Findings• Micropropagation through in vitro shoot bud culture hasbeen developed in guava cv. Allahabad Safeda.

• 3 cm long nodal segments were surface sterilized usingCarbendazime 0.1% and PVP 100 mg/l.

• Explants were further treated with HgCl2 0.1% for 5minutes aseptically, followed by six washing in steriledistilled water.

• Quick shoot bud induction was achieved within five days inMS medium supplemented with BAP 3.0 mg/l.

• The proliferation of microshoots (2.67 shoots/explant) wasachieved in same medium (MS+BAP 3.0 mg/l).

• Rooting was achieved in MS medium containing IBA 10mg/l within 17.6 days.

• In vitro rooted plants were acclimatized on coconut huskcontaining ½ strength MS plant salt mixture and undershade net house (50 % shade 70-80 % RH).

• Around 88.48% plants were survived duringacclimatization.

• Acclimatized plantlets of guava cv. Allahabad Safeda wereplanted in field during rainy season.

ED

CBA

F

Fig: Micropropagation of Psidium guajava (Guava). (A) In vitro shoot budinduction, (B) proliferation, (C) Rooting, (D) Acclimatization, (E) Acclimatizedplants in polyhouse (F) Plant growing in field

Emblica officinallis Gaertn.

Introduction

• Indian gooseberry or aonla (Emblica officinalis Gaertn.)belongs to the family euphorbiaceae and is well known forits medicinal and therapeutic properties.

• It is a rich source of Vit C and used in fruit processingindustries.

• The use of micropropagation approach for accelerating theproduction of clonal stock of commercial cultivars in Indian.

Findings• 3 cm long nodal segments were surface sterilized usingCarbendazime 0.1% and PVP 100 mg/l.

• Explants were further treated with HgCl2 0.1% for 8minutes aseptically, followed by six washing in steriledistilled water.

• The maximum survival (66.49%) and minimumcontamination (33.51%) of shoot buds were observedduring surface sterilization.

• MS medium supplemented with Kinetin 13.9 3 +GA3 4.33+Glutamine 342.11 mM gave the early bud break (7days)and highest shoot proliferation (13.33 shoots/culture).

• 2-3 cm long microshoots were cultured on ½ MS+ IBA49.20 mM+ NAA 10.74 mM and activated charcoal 0.1%,in this treatment 4 roots/explant was achieved in 7.33 days.

• In vitro rooted plants were acclimatized onSoil+Sand+FYM (1:1:1) containing ½ MS plant saltmixture containing paclobuterazole 1.0 mg/l under shadenet house (50 % shade 70-80 % RH).

• 72.22% plants were survived during acclimatization.

• Acclimatized plants were successfully transferred to fieldduring rainy season.

ED

CBA

F

Fig: Micropropagation of Emblica officinalis (Aonla). (A) In vitro shoot budinduction, (B‐C) proliferation, (D) Rooting, (E) Acclimatization, (F)Acclimatized plants in polyhouse

Syzygium cuminii Skeels.

Introduction• Jamun or black plum (family myrtaceae) is an importantindigenous underutilized fruits of commercial value.

• It is good source of iron and anti-diabetic compounds.

• The seeds are claimed to contain alkaloid, jambosine,and glycoside jambolin or antimellin, which halts thediastatic conversion of starch into sugar and seedextract has lowered blood pressure.

• Jamun orcharding can help in the management ofwastelands.

• In order to produce large scale true-to-type plantingmaterial, micropropagation can be gainfully employed.

Findings• The shoot buds were taken from eight years old plant forprotocol establishment.

• 3 cm long nodal segments were surface sterilized usingCarbendazime 0.1% and PVP 100 mg/l followed bytreatment with HgCl2 0.1% for 6 minutes aseptically,followed by six washing in sterile distilled water.

• Nodal segments were cultured on MS medium +BAP 2.0mg/l. This treatment gave earlier shoot bud induction in just5.6 days.

• Highest shoot proliferation was found in MS medium+BAP2.0 mg/l+ GA3 0.5 mg/l+Casein hydrosylate 150 mg/l.

• Microshoots were rooted cent percent on ½ MS+ IBA 5mg/l+Activated charcoal 100 mg/l in just 12 days.

• In vitro rooted plants were acclimatized onSoil+Sand+FYM (1:1:1) containing ½ MS plant saltmixture fortifird with paclobuterazole 1.0 mg/l undershade net house (50 % shade 70-80 % RH).

• 80% plants were survived during acclimatization.

• After three month of acclimatization plants were transferto field for further analysis.

BA

D

C

E

Fig: Micropropagation of Syzygium cuminii Skeels.(Jamun). (A) In vitroshoot bud induction, (B) Culture establishment (C) Proliferation inmicroshoots, (D) Rooting, (E) Acclimatization, (F) Acclimatized plantgrowing in field.

Mangifera indica L.

Introduction• Mango ( Mangifera indica L.) is the most important fruit cropbecause of its wide adaptability, high nutritive value, richnessin variety, delicious taste, excellent flavour, attractiveappearance and commercial utility in India as well as in manypart of the world.

• Conventional breeding in perennial crops is difficult and timeconsuming.

• A standard uniform protocol of regeneration is the prime andforemost prerequisite for not only improve the productivity, theproduction from the existing area but also development oftransgenics in mango for various traits.

Findings• Nucellar embryogenesis was induced in differentmonoembryonic and polyembryonic cultivars of mango(Mangifera indica L) viz., Dashehari, Amrapali, Bapakkai,Kurukkan and Moovandan.

• Nucellus tissue excised from 3.5 cm long fruits of thesecultivars developed pro-embryonic calli on modified MSmedium supplemented with 2,4-D 4.52μM + malt extract0.05% and spermidine 13.78μM. • Among all the cultivars, polyembryonic cultivars gave higherlevel of somatic embryogenesis in comparison tomonoembryonic.

• Among all polyembryonic cultivars. Bappakai produced 187.33embryos per explant followed by Kurukkan 158.33 embryos perexpalnt and Movandan 146.45 embryos per explant, where asmonoembryonic cultivars shows comparatively lower level ofsomatic embryogenesis. • Dashehari gave rise to 97.25 embryos per explant followed byAmrapali 82.33 embryos in 100 days under dark cultureconditions.

• However, all the differentiated embryos proliferated on mediumhaving low level of sucrose (4% w/v) and auxin (2, 4-D 2.26μM).

• Most of the proembryonic calli converted into heart shaped andcotyledonary embryos by reducing temperature to 15oC.

• Somatic embryos were matured on modified MS mediumfortified with ABA 0.38μM+ IAA 0.57μM and PEG30.30μM.

• Matured somatic embryos germinated on MS mediumsupplemented with NAA 2.68μM+ kinetin 11.60μM andglutamine 2736.9μM.

• Among all cultivars, Bappakai showed higher germination(39.33) followed by Kurukkan (29.97 %), Movandan(28.25%), Deshahari (26.45%) and Amrapali (25.25%).

Monoembryonic cultivars of mango

Bapakkai Kurukkan Moovandan

Dashehari Amrapali

Polyembryonic cultivars of mango

Different stages of somatic embryogenesis in mango

CBA

FEDDifferent stages of somatic embryogenesis in mango (Mangifera indica L).A-B Somatic embryo induction from nucellar tissues, C-Proliferation and development ofglobular embryos, D- conversion of SE into heart shaped, E- early cotyledonary stage, F-late cotyledonary shaped embryo.

Cont…

G H

I

Different stages of somatic embryogenesis in mango

Different stages of somatic embryogenesis in mango (Mangifera indica L). G-rooting and conversion into early stage of plants and H-rooted plants growingvigorously in liquid culture medium and I- plant in polyhouse during acclimatization.

Shoot tip transformation of papaya 

(Carica papaya L.)

Introduction• Papaya (Carica papaya L.) is a popular and economically 

important and medicinal fruit tree of tropical and subtropical countries.

• It has varied uses in the beverage, food and pharmaceutical industries, in chill‐proofing beer, tenderizing meat, drug preparations for digestive ailments and treatment of gangrenous wounds. 

• Papaya is host to various species of pests and pathogens and affected by various viral, fungal and nematode diseases.

• Here we are reporting, Development of transgenic papayaresistant to Papaya Leaf Curl Virus (PLCuV) and PapayaRingspot Virus (PRSV) using A. tumifaciens.

Findings• In vitro seedling plants were raised from immature zygoticembryos excised from 90-100 days old white, plumpimmature seeds of Pusa Delicious in ½ MS mediumcontaining BAP 0.2 mg/l+ MS Vitamins 1.0 ml/l (extra),agar 0.8% and sucrose 3%.

• A. tumifaciens was grown in LB medium containing 50mg/l Kanamycin in the dark at 28oC in incubator shaker at120 rpm for 24 hrs (1.0 OD at OD600).

• One week old Shoot tips (0.5cm), Carborandumwounding, 30 min infection period, 72 hrs co-cultivationperiod, Cefotaxime 500 mg/l, Acetosyringone 100 µMand Spermidine 1.0 mM gave higher (8.8%) putativetransformats.

• Plants were rooted on ½ MS medium containing IBA 3.0mg/l.

• Transformed plants were diagnosed using forward andreverse primers of cp (410 bp), rep (479 bp) and npt II(480 bp) PCR reactions.

Fig: Papaya shoot tip transformation using Agrobacterium tumifaciens. (A) 0.5 cm long shoot tipas an explant, (B) Selection and regeneration of transformed shoot tip using Kanamycin 150 mg/lafter 12 weeks, (C) Rooting in transformed papaya shootlets using IBA 3.0 mg/l, (D)Acclimatization of PCR positive plantlet, (E) Acclimatized plants are under evaluation inTransgenic glass house, (F) Gel picture showing 480 bp npt II bands.

Publications• Pati R, Mishra M, Chandra R and Muthukumar M (2013). Histological andbiochemical changes in Aegle marmelos Corr. before and after acclimatization.Tree Genetics and Molecular Breeding. 3(3): 12-18.

• Pati R, Chandra R, Chauhan UK, Mishra M and Srivastiva N (2008). In vitroclonal propagation of bael (Aegle marmelos Corr.) cv. CISHB1 throughenhanced axillary branching. Physiology and Molecular Biology of Plants.14(4): 337-346.

• Pati R, Chandra R, Chauhan UK and Mishra M (2008). In vitro plantregeneration from mature explant of Aegle marmelos Corr.) CV. CISH-B2.Science and Culture. 74(9-10): 359-367.

• Mishra, M. Chandra, R., Pati, R. and Bajpai, A. (2007). Micropropagation ofGuava (Psidium guajava L.). Acta Horticulturae, 735: 155-158.

• Mishra M, Pati R and Chandra R (2006). Clonal micropropagation of Indiangooseberry (Emblica officinalis Gaertn). Indian Journal of Genetics and PlantBreeding. 66(4): 361-361.

• Mishra M, Chandra R, Pati R (2008). In vitro regeneration and genetic fidelitytesting of Aegle marmelos (Corr.) plants. Indian Journal of Horticulture. 65(1):6-11.

• Mishra M, Pati R, Chandra R. et al. R (2010). Micropropagation of Mangiferaindica L. cv. Kurakkan through somatic embryogenesis. Indian Journal ofGenetics and Plant Breeding. 70(1): 85-90.

• Mishra M, Chandra R, Pati R, Jain RK and Agarawal S (2010). Shoot tiptransformation of papaya (Carica papaya L.). Acta Horticulturae, 851: 219-226.

• Mishra M, Chandra R, Tiwari RK, Pati R and Pathak RK (2005).Micropropagation of certain under utilized fruit crops: A Review. Small fruitsReview. 4(4): 7-18.

• Pati R. and Muthukumar M. (2013). Genetic transformation in Aegle marmrlosCorr. In: Biotechnology of neglected and underutilized crops, edited by S.M.Jain and S. Dutta Gupta. Springer . p.343-365. [ISBN: 978-94-007-5500-0].

• Chandra R, Pati R and Mishra M (2010). Mango. In: Advances in HorticulturalBiotechnology Vol.-1 Regeneration Systems- Perennial Fruit Crops and Spices.Eds., Singh HP, Parthasarathy VA and Nirmal Babu K. Westville PublishingHouse, New Delhi, pp. 73-90. [ISBN-978-11-85873-65-7].

• Mishra M, Pati R and Chandra R (2009). “Clonal micropropagation ofsubtropical fruit trees”. In: Forest Biotechnology in India, edited by MandalAK, Ansari SA and Narayanan C. Vedams eBooks (P) Ltd., New Delhi, India.[ISBN-81-89304-56-9].

• Pati R, Gupta VK, Yadava LP, Srivastiva N, Gupta A and Modi DR (2008).“Agrobacterium- As Natural Tool for Plant Genetic Engineering”: In: PotentialMicroorganism for Sustainable Agriculture, edited by Prof. D.K. Maheshwariand R.C. Dubey, I.K. International Pvt. Ltd., New Delhi, pp.436-459. [ISBN-8190746205].

Thank you