Micropropagation is a proven means of producing millions of identical plants under a controlled and aseptic condition, independent of seasonal constraints. It not only provides economy of time and space but also gives greater output and allows further augmentation of elite disease free propagules.India is homeland of many important fruit crops such as Indian gooseberry (Emblica officinalis Gaertn), bael (Aegle marmelos Corr.), Guava (, Psidium guajava), jamun or black plum (Syzygium cuminii L. Skeels.), Mango (Mangifera indica) and Papaya (Carica papaya).

1. Micropropagation in
fruit crops
Rajesh Pati PhD,
Maneesh Mishra and Ramesh Chandra
e‐mail: rajeshpati777@gmail.com
Central Institute for Subtropical Horticulture,
Lucknow, India

2. Introduction
• Micropropagation is a proven means of producing millions
of identical plants under a controlled and aseptic condition,
independent of seasonal constraints.
• It not only provides economy of time and space but also
gives greater output and allows further augmentation of elite
disease free propagules.
• India is homeland of many important fruit crops such as
Indian gooseberry (Emblica officinalis Gaertn), bael (Aegle
marmelos Corr.), Guava (, Psidium guajava), jamun or
black plum (Syzygium cuminii L. Skeels.), Mango
(Mangifera indica) and Papaya (Carica papaya).

3. • Most of these crops have medicinal value and are suitable
for growing under marginal situations.
• The commercial production of these crops is restricted due
to the shortage of desirable planting material.
• Micropropagation can play an important role in rapidly
increasing new cultivars of these fruit crops.

4. Aegle marmelos Corr.

5. Introduction
• Aegle marmelos Corr., belongs to family Rutaceae, is more
prized for its pharmacological virtues than its edible quality.
• Because of pharmacological importance, it’s become potential
candidate for developing transgenics to enhance its medicinal
properties.
• Maximum mortality of micropropagated plants occur during
acclimatization phase because plantlets undergo rapid and
extreme changes in physiological functioning, histological
and biochemical changes.
• In order to investigate the actual reason of this limitation, test
samples were collected at different micropropagation stages
(In vitro, and acclimation).

6. Findings
• Rapid clonal micropropagation protocol of Aegle marmelos Corr.
cv. CISH-B1 and CISH-B2 was achieved by nodal stem segment
of mature bearing tree.
• Three centimeter long shoots having one axillary bud excised
from 10-15th nodal region of shoots during September gave quick
in vitro bud burst (5.33 days) when cultured on MS medium
supplemented with BAP, 8.84 μM + IAA 5.7 μM.
• The maximum number of proliferated shoots (9.0/explant) were
obtained on same medium supplemented with BAP 8.84 μM +
IAA 5.7 μM.
• The micro shoots were rooted (100 %) on ½ strength MS medium
supplemented with IBA 49.0 + IAA 5.7 μM. .

7. • In vitro rooted plants were acclimatized on coconut husk
containing ½ strength MS plant salt mixture and under shade
net house (50 % shade 70-80 % RH).
• The plants were established in the field after acclimatization.
• The micropropagated plants were tested for its genetic fidelity
using 13 RAPD, 3 ISSR and 2 DAMD primers.
• Profile obtained by all the three Single Primer Amplification
Reaction (SPAR) technique from mother tree and
micropropagated plants revealed genetic uniformity of
micropropagated plants with that of mother tree.

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

9. Genetic uniformity

10. Histological and biochemical changes
• Maximum mortality of micropropagated plants occur during
acclimatization phase, because plantlets undergo rapid and
extreme changes in physiological functioning, histological
and biochemical changes.
• In order to investigate the actual reason of this limitation, test
samples (Leaf, stem and root) were collected at different
stages of micropropagated plants (In vitro and acclimation).
• The biochemical result showed that micropropagated plantlets
produced significantly low total chlorophyll (0.042 mg/g fresh
weight), reducing sugar (3.227%), NR activity (1.353
NO2/h/g fresh weight) and but higher protein (0.048 μg/g)
during in vitro phase.

11. • The in vitro raised plants showed abnormal histological features
like altered leaf mesophyll, absence of thick cuticle, sunken
stomata, poorly developed stem and root histology.
• Photoautrophic mode of nutrition during in vitro phase increased
the survival rate during acclimatization compared to
photoheterotrophic mode of nutrition
• Photoautotrophism phenemoneon has substantial influence on
the physiology and development of in vitro regenerated Aegle
marmelos Corr. plantlets.

12. Anatomy of leaf
A
B
(A) Whole mount of in vitro leaf showed open type stomata with fully turgid
guard cells, (B) whole mount of acclimatized leaf showed partially
closed stomata.

13. Anatomy of leaf
A
B
(A) T.S. of in vitro leaf, showed single layered epidermis with almost no cuticle and
single layered and poorly developed palisade parenchyma with poorly
developed vascular tissue.
(B) T.S. of acclimatized leaf, showed single layered epidermis with thick cuticle and
well developed double layered palisade mesophyll while the lower side had
spongy parenchyma with air spaces and sunken type stomata.

14. Anatomy of stem
A
B
(A) T.S. of in vitro stem, had a polystelic structures, vascular bundles were arranged
in a ring and they were conjoint collateral and open no corck cambium, showed
primary medullary rays, uniseriate epidermis, no cuticle, distinct endoderm was
absent while cortex was parenchymatous.
(B) T.S. of acclimatized stem, well developed cork cambium, parenchymatous pith
but the pith was mucilaginous, distinct secondary growth with well developed
wood and large woody vessels, epidermis was uniseriate and covered with cuticle,
and cortex was collenchymatous.

15. Anatomy of root
A
B
(A) T.S. of in vitro root, showed poorly developed pith, undifferentiated cortex with
very less amount of storage tissue and no secondary growth at all.
(B) T.S. of acclimatized Root, had a well developed parenchymatous cortex with tannin
cells, pericycle is made of stone cells vascular cylinder consisted of secondary
xylem towards inner side and secondary phloem towards outer side, secondary
xylem showed wide vessels scattered among trachieds, medullary rays were present
while pith was negligible.

16. Psidium guajava (L.)

17. Introduction
• Guava (Psidium guajava Linn.) commonly known for its
food, nutraceutical and commercial values throughout
the world.
• The guava plant parts are used for the development of
various industrial and pharmaceutical products.
• Guava contains broad spectrum of phytochemicals
including polysaccharides, vitamins, essential oils,
minerals, enzymes, proteins, sesquiterpenoid alcohols
and triterpenoid acids , alkaloids, glycosides, steroids,
flavanoids, tannins, saponins .
• Guava is very rich in antioxidants and vitamins and also
high in lutein, zeaxanthine and lycopene .

18. Findings
• Micropropagation through in vitro shoot bud culture has
been developed in guava cv. Allahabad Safeda.
• 3 cm long nodal segments were surface sterilized using
Carbendazime 0.1% and PVP 100 mg/l.
• Explants were further treated with HgCl2 0.1% for 5
minutes aseptically, followed by six washing in sterile
distilled water.
• Quick shoot bud induction was achieved within five days in
MS medium supplemented with BAP 3.0 mg/l.
• The proliferation of microshoots (2.67 shoots/explant) was
achieved in same medium (MS+BAP 3.0 mg/l).

19. • Rooting was achieved in MS medium containing IBA 10
mg/l within 17.6 days.
• In vitro rooted plants were acclimatized on coconut husk
containing ½ strength MS plant salt mixture and under
shade net house (50 % shade 70-80 % RH).
• Around 88.48%
acclimatization.
plants
were
survived
during
• Acclimatized plantlets of guava cv. Allahabad Safeda were
planted in field during rainy season.

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

21. Emblica officinallis Gaertn.

22. Introduction
• Indian gooseberry or aonla (Emblica officinalis Gaertn.)
belongs to the family euphorbiaceae and is well known for
its medicinal and therapeutic properties.
• It is a rich source of Vit C and used in fruit processing
industries.
• The use of micropropagation approach for accelerating the
production of clonal stock of commercial cultivars in Indian.

23. Findings
• 3 cm long nodal segments were surface sterilized using
Carbendazime 0.1% and PVP 100 mg/l.
• Explants were further treated with HgCl2 0.1% for 8
minutes aseptically, followed by six washing in sterile
distilled water.
• The maximum survival (66.49%) and minimum
contamination (33.51%) of shoot buds were observed
during 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).

24. • 2-3 cm long microshoots were cultured on ½ MS+ IBA
49.20 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 on
Soil+Sand+FYM (1:1:1) containing ½ MS plant salt
mixture containing paclobuterazole 1.0 mg/l under shade
net house (50 % shade 70-80 % RH).
• 72.22% plants were survived during acclimatization.
• Acclimatized plants were successfully transferred to field
during rainy season.

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

26. Syzygium cuminii Skeels.

27. Introduction
•
Jamun or black plum (family myrtaceae) is an important
indigenous 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 the
diastatic conversion of starch into sugar and seed
extract has lowered blood pressure.
•
Jamun orcharding can help in the management of
wastelands.
•
In order to produce large scale true-to-type planting
material, micropropagation can be gainfully employed.

28. Findings
• The shoot buds were taken from eight years old plant for
protocol establishment.
• 3 cm long nodal segments were surface sterilized using
Carbendazime 0.1% and PVP 100 mg/l followed by
treatment with HgCl2 0.1% for 6 minutes aseptically,
followed by six washing in sterile distilled water.
• Nodal segments were cultured on MS medium +BAP 2.0
mg/l. This treatment gave earlier shoot bud induction in just
5.6 days.
• Highest shoot proliferation was found in MS medium+BAP
2.0 mg/l+ GA3 0.5 mg/l+Casein hydrosylate 150 mg/l.

29. • Microshoots were rooted cent percent on ½ MS+ IBA 5
mg/l+Activated charcoal 100 mg/l in just 12 days.
• In vitro rooted plants were acclimatized on
Soil+Sand+FYM (1:1:1) containing ½ MS plant salt
mixture fortifird with paclobuterazole 1.0 mg/l under
shade net house (50 % shade 70-80 % RH).
• 80% plants were survived during acclimatization.
• After three month of acclimatization plants were transfer
to field for further analysis.

30. A
B
D
E
C
Fig: Micropropagation of Syzygium cuminii Skeels.(Jamun). (A) In vitro
shoot bud induction, (B) Culture establishment (C) Proliferation in
microshoots, (D) Rooting, (E) Acclimatization,
(F) Acclimatized plant
growing in field.

31. Mangifera indica L.

32. Introduction
• Mango ( Mangifera indica L.) is the most important fruit crop
because of its wide adaptability, high nutritive value, richness
in variety, delicious taste, excellent flavour, attractive
appearance and commercial utility in India as well as in many
part of the world.
• Conventional breeding in perennial crops is difficult and time
consuming.
• A standard uniform protocol of regeneration is the prime and
foremost prerequisite for not only improve the productivity, the
production from the existing area but also development of
transgenics in mango for various traits.

33. Findings
• Nucellar embryogenesis was induced in different
monoembryonic 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 these
cultivars developed pro-embryonic calli on modified MS
medium supplemented with 2,4-D 4.52μM + malt extract
0.05% and spermidine 13.78μM.
• Among all the cultivars, polyembryonic cultivars gave higher
level of somatic embryogenesis in comparison to
monoembryonic.

34. • Among all polyembryonic cultivars. Bappakai produced 187.33
embryos per explant followed by Kurukkan 158.33 embryos per
expalnt and Movandan 146.45 embryos per explant, where as
monoembryonic cultivars shows comparatively lower level of
somatic embryogenesis.
• Dashehari gave rise to 97.25 embryos per explant followed by
Amrapali 82.33 embryos in 100 days under dark culture
conditions.
• However, all the differentiated embryos proliferated on medium
having low level of sucrose (4% w/v) and auxin (2, 4-D 2.26μM).
• Most of the proembryonic calli converted into heart shaped and
cotyledonary embryos by reducing temperature to 15oC.

35. • Somatic embryos were matured on modified MS medium
fortified with ABA 0.38μM+ IAA 0.57μM and PEG
30.30μM.
• Matured somatic embryos germinated on MS medium
supplemented with NAA 2.68μM+ kinetin 11.60μM and
glutamine 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%).

36. Monoembryonic cultivars of mango
Dashehari
Amrapali
Polyembryonic cultivars of mango
Bapakkai
Kurukkan
Moovandan

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

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

39. Shoot tip transformation of
papaya
(Carica papaya L.)

40. 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 papaya
resistant to Papaya Leaf Curl Virus (PLCuV) and Papaya
Ringspot Virus (PRSV) using A. tumifaciens.

41. Findings
• In vitro seedling plants were raised from immature zygotic
embryos excised from 90-100 days old white, plump
immature seeds of Pusa Delicious in ½ MS medium
containing 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 50
mg/l Kanamycin in the dark at 28oC in incubator shaker at
120 rpm for 24 hrs (1.0 OD at OD600).
•
One week old Shoot tips (0.5cm), Carborandum
wounding, 30 min infection period, 72 hrs co-cultivation
period, Cefotaxime 500 mg/l, Acetosyringone 100 µM
and Spermidine 1.0 mM gave higher (8.8%) putative
transformats.

42. • Plants were rooted on ½ MS medium containing IBA 3.0
mg/l.
• Transformed plants were diagnosed using forward and
reverse primers of cp (410 bp), rep (479 bp) and npt II
(480 bp) PCR reactions.

43. Fig: Papaya shoot tip transformation using Agrobacterium tumifaciens. (A) 0.5 cm long shoot tip
as an explant, (B) Selection and regeneration of transformed shoot tip using Kanamycin 150 mg/l
after 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 in
Transgenic glass house, (F) Gel picture showing 480 bp npt II bands.

44. Publications
•
Pati R, Mishra M, Chandra R and Muthukumar M (2013). Histological and
biochemical 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 vitro
clonal propagation of bael (Aegle marmelos Corr.) cv. CISHB1 through
enhanced axillary branching. Physiology and Molecular Biology of Plants.
14(4): 337-346.
•
Pati R, Chandra R, Chauhan UK and Mishra M (2008). In vitro plant
regeneration 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 of
Guava (Psidium guajava L.). Acta Horticulturae, 735: 155-158.
•
Mishra M, Pati R and Chandra R (2006). Clonal micropropagation of Indian
gooseberry (Emblica officinalis Gaertn). Indian Journal of Genetics and Plant
Breeding. 66(4): 361-361.

45. • Mishra M, Chandra R, Pati R (2008). In vitro regeneration and genetic fidelity
testing 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 Mangifera
indica L. cv. Kurakkan through somatic embryogenesis. Indian Journal of
Genetics and Plant Breeding. 70(1): 85-90.
• Mishra M, Chandra R, Pati R, Jain RK and Agarawal S (2010). Shoot tip
transformation of papaya (Carica papaya L.). Acta Horticulturae, 851: 219226.
• Mishra M, Chandra R, Tiwari RK, Pati R and Pathak RK (2005).
Micropropagation of certain under utilized fruit crops: A Review. Small fruits
Review. 4(4): 7-18.
• Pati R. and Muthukumar M. (2013). Genetic transformation in Aegle marmrlos
Corr. 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].

46. • Chandra R, Pati R and Mishra M (2010). Mango. In: Advances in Horticultural
Biotechnology Vol.-1 Regeneration Systems- Perennial Fruit Crops and Spices.
Eds., Singh HP, Parthasarathy VA and Nirmal Babu K. Westville Publishing
House, New Delhi, pp. 73-90. [ISBN-978-11-85873-65-7].
• Mishra M, Pati R and Chandra R (2009). “Clonal micropropagation of
subtropical fruit trees”. In: Forest Biotechnology in India, edited by Mandal
AK, 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: Potential
Microorganism for Sustainable Agriculture, edited by Prof. D.K. Maheshwari
and R.C. Dubey, I.K. International Pvt. Ltd., New Delhi, pp.436-459. [ISBN8190746205].

47. Thank you