Micropropagation in fruit crops

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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).

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Micropropagation in fruit crops

  1. 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. 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. 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. 4. Aegle marmelos Corr.
  5. 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. 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. 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. 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. 9. Genetic uniformity
  10. 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. 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. 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. 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. 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. 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. 16. Psidium guajava (L.)
  17. 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. 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. 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. 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. 21. Emblica officinallis Gaertn.
  22. 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. 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. 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. 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. 26. Syzygium cuminii Skeels.
  27. 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. 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. 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. 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. 31. Mangifera indica L.
  32. 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. 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. 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. 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. 36. Monoembryonic cultivars of mango Dashehari Amrapali Polyembryonic cultivars of mango Bapakkai Kurukkan Moovandan
  37. 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. 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. 39. Shoot tip transformation of  papaya  (Carica papaya L.)
  40. 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. 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. 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. 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. 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. 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. 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. 47. Thank you

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