Protocols for Micropropagation of Woody Trees and Fruits

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336 R.I. IYER compounds (Iyer et al., 2004, 2005a,b) of potent clinical value, some of which had not been reported earlier in vivo. Elicitation of the production of anthraquinones in vitro by chitosan was obtained (Iyer et al., 2005b). There was strong anti-bacterial activity against the human pathogens Salmonella typhi and Staphylococcus aureus (Iyer et al., 2005a) in the spent medium of the embryogenic cultures and nucleic acid base analogues could be detected in it. This reveals the potential of the embryogenic cultures for continuous production of valuable bioactive phytochemicals. The development of in vitro propagation techniques is done with the culture of different explants including zygotic embryos, leaf segments, nodal explants, and stem segments. 2. EXPERIMENTAL PROTOCOL 2.1. In vitro Propagation from Zygotic Embryos Zygotic embryos have been used as explants in many woody plants since these are relatively contaminant-free being enclosed in the fruit and seed tissues. Zygotic embryos of nutmeg are surrounded by the massive endosperm. Somatic embryogenesis was obtained from zygotic embryos in media with activated charcoal (Iyer et al., 1994, 2000). The extent of somatic embryogenesis was dependent on the size of the zygotic embryos. 2.1.1. Explant Preparation The zygotic embryos from ripe, freshly-harvested nutmeg fruits were used as explants. Growing conditions of mother plants. The fruits were obtained from State Horticultural Farm, Kallar (11.18° N, 76.5° E), Coimbatore, India with rainfall of 150–200 cm well distributed throughout the year. The soil is well-drained, deep, loamy and rich in organic matter. Explant excision and sterilization 1. Fully ripe split fruits (about 5 cm diameter) were harvested from the trees. 2. Husk of the fruit was removed. 3. Seed was then freed from the surrounding aril. All further operations were carried out in a laminar flow cabinet. 4. Seeds were swabbed with ethanol. 5. They were surface-sterilized by immersing in 0.1% HgCl2 for 3 min, followed by several rinses with sterile distilled water. 6. Shell was removed to facilitate embryo removal. 7. Embryos were excised with a sharp scalpel and a pair of forceps. 8. The intact zygotic embryos and the pieces of zygotic embryos fragmented during excision were used as explants.
IN VITRO PROPAGATION OF NUTMEG Table 1. Concentration (mg/L ) of inorganic salts in the basal media. Constituents Ammonium nitrate Medium A 400 Media B1– B 7 1650.0 Boric acid 6.2 6.2 Calcium chloride, anhydrous 332.2 332.2 Cobalt chloride,6 H2O 0.025 0.025 Cupric sulphate, 5 H 2O 0.025 0.025 Disodium EDTA 74.5 37.26 Ferrous sulphate, 7 H2O 55.7 27.8 Magnesium sulphate 180.7 — Magnesium sulphate, 7 H2O — 370.0 Manganese sulphate, H2O 16.9 16.9 Potassium iodide 0.3 0.83 Potassium nitrate 480.0 1900.0 Potassium phosphate, monobasic — 170.0 Sodium molybdate, 2 H2O 0.25 0.25 Sodium phosphate, monobasic, anhydrous 330.6 — Zinc sulphate, 7 H2O 8.6 8.6 Table 2. Organics, growth regulators (mg/L) and other supplements added to the different inductive media. Components (mg/L) A* B1 B2 B3 B4 B5 B6 B7 Myo-inositol 1000 100 100 100 100 100 100 100 Sucrose 20000 30000 30000 30000 30000 30000 30000 30000 Thiamine HCl 8.5 10 0.5 0.5 0.5 0.5 0.5 0.5 Nicotinic acid 5 1 0.4 0.4 0.4 0.4 0.4 0.4 Pyridoxine 5 1 0.5 0.5 0.5 0.5 0.5 0.5 HCl 2,4-D 10 1 4 — — — — — IAA 10 1 2 — — — — — NAA — — — — 5 5 5 5 GA3 — — — 5 — — 1 — BAP — 4 — — 1 1 — 1 Kinetin 10 — — — — — — — TDZ — — — — — 0.1 — 0.05 Activated charcoal 250 250 250 250 250 250 250 250 PEG 4000 — — — — — — 50,00 — * To medium A , 10% (w/ v) potato extract was added. Potato extract was prepared from 100 g potato boiled in water, mashed, cooled and filtered. Abbreviations. 2,4-D: 2,4 dichlorophenoxyacetic acid; IAA: indole-3-acetic acid; NAA: naphthaleneacetic acid; BAP: benzylaminopurine; GA3 : gibberellic acid; TDZ: thidiazuron; PEG: polyethylene glycol. 0 337
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PROTOCOLS FOR MICROPROPAGATION OF W
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A C.I.P. Catalogue record for this
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vi TABLE OF CONTENTS 14. Root induc
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viii TABLE OF CONTENTS 43. Micropro
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x PREFACE on precise stepwise proto
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CHAPTER 1 TOTIPOTENCY AND THE CELL
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TOTIPOTENCY AND THE CELL CYCLE 5 a
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2.2. Plant Bioregulators and the Ce
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TOTIPOTENCY AND THE CELL CYCLE 9 in
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TOTIPOTENCY AND THE CELL CYCLE 11 F
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Bartel, D. (2004) MicroRNAs: genomi
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CHAPTER 2 MICROPROPAGATION VIA ORGA
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MICROPROPAGATION OF SLASH PINE Tabl
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MICROPROPAGATION OF SLASH PINE Amon
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2.6. Field Testing MICROPROPAGATION
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CHAPTER 3 MICROPROPAGATION OF COAST
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MICROPROPAGATION OF SEQUOIA SEMPERV
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CHAPTER 4 MICROPROPAGATION OF PINUS
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MICROPROPAGATION OF PINUS PINEA L.
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42 2.1. Organ Culture K. ISHII ET A
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44 K. ISHII ET AL. scent light, 70
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46 Chemicals (mg/l) K. ISHII ET AL.
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48 Table 3. Effects of plant growth
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50 K. ISHII ET AL. Gupta, P.K. & Du
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52 C. HARGREAVES AND M. MENZIES (Me
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54 C. HARGREAVES AND M. MENZIES Con
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56 2.1.3. Organogenesis from Field-
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58 C. HARGREAVES AND M. MENZIES Fig
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60 C. HARGREAVES AND M. MENZIES Fig
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62 C. HARGREAVES AND M. MENZIES For
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64 C. HARGREAVES AND M. MENZIES and
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CHAPTER 7 GENETIC FIDELITY ANALYSES
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PLANT GENETIC FIDELITY 69 Plant mat
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e added to samples, as this fluoroc
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11. Determine the mean channel numb
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3. In addition to testing various b
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GeneScan internal size standards: 4
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3500 3000 2500 2000 1500 1000 500 0
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PLANT GENETIC FIDELITY 81 microprop
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PLANT GENETIC FIDELITY 83 Steinkell
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86 2.1. Explant Preparation M.G. OS
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88 WPM (Lloyd & McCown, 1980) Macro
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90 M.G. OSTROLUCKÁ ET AL. on the m
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CHAPTER 9 MICROPROPAGATION OF MEDIT
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2.1. Explant Preparation MICROPROPA
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MICROPROPAGATION OF MEDITERRANEAN C
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108 D.T. NHUT ET AL. Larue (1953) a
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110 D.T. NHUT ET AL. HgCl2 added wi
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112 2.4. Establishment of Shoot Cul
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114 D.T. NHUT ET AL. Table 5. Effec
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116 D.T. NHUT ET AL. culture to be
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118 D. EWALD ability of the plant m
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120 D. EWALD Figure 1. Yew micropro
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122 D. EWALD Root development. Afte
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CHAPTER 12 MICROPROPAGATION OF LARI
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MICROPROPAGATION OF LARIX SPECIES V
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CHAPTER 13 PROPAGATION OF SELECTED
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PROPAGATION OF SELECTED PINUS GENOT
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CHAPTER 14 ROOT INDUCTION OF PINUS
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ROOT INDUCTION OF PINUS SYLVESTRIS
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ROOT INDUCTION OF PINUS SYLVESTRIS
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CHAPTER 15 MICROPROPAGATION OF BETU
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MICROPROPAGATION OF BETULA PENDULA
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CHAPTER 16 PROTOCOL FOR DOUBLED-HAP
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DOUBLED-HAPLOID MICROPROPAGATION IN
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CHAPTER 17 IN VITRO PROPAGATION OF
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IN VITRO PROPAGATION OF FRAXINUS SP
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Axillary shoots (number) IN VITRO P
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CHAPTER 18 MICROPROPAGATION OF BLAC
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MICROPROPAGATION OF BLACK LOCUST 19
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2.5. Rooting and Acclimatization MI
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MICROPROPAGATION OF BLACK LOCUST 19
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202 V. RAJESWARI AND K. PALIWAL tha
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204 V. RAJESWARI AND K. PALIWAL Mak
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206 V. RAJESWARI AND K. PALIWAL 2.3
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208 V. RAJESWARI AND K. PALIWAL Fig
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210 2.8. Hardening V. RAJESWARI AND
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CHAPTER 20 MICROPROPAGATION OF SALI
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MICROPROPAGATION OF SALIX CAPREA L.
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CHAPTER 21 MICROPROPAGATION OF CEDR
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2.1. Establishment of Shoot Culture
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MICROPROPAGATION OF CEDRELA FISSILI
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238 programmes is somatic embryogen
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240 Figure 2. A) Induction of organ
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242 2.4.1. Rooting of Apical and Ba
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244 of donor individuals and/or by
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246 J. MALÀ ET AL. Karnosky, D.F.,
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CHAPTER 23 MICROGRAFTING IN GRAPEVI
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MICROGRAFTING IN GRAPEVINE 251 and
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MICROGRAFTING IN GRAPEVINE 253 Tabl
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2.4. Culture Conditions MICROGRAFTI
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MICROGRAFTING IN GRAPEVINE 257 Feuc
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CHAPTER 24 MICROGRAFTING GRAPEVINE
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MICROGRAFTING GRAPEVINE FOR VIRUS I
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CHAPTER 25 APRICOT MICROPROPAGATION
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APRICOT MICROPROPAGATION Figure 1.
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APRICOT MICROPROPAGATION Figure 2.
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APRICOT MICROPROPAGATION 2.2.2. Hyp
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2.4. Acclimatization APRICOT MICROP
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APRICOT MICROPROPAGATION occurs fre
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CHAPTER 26 IN VITRO CONSERVATION AN
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IN VITRO CONSERVATION AND MICROPROP
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IN VITRO CONSERVATION AND MICROPROP
Page 284 and 285: IN VITRO CONSERVATION AND MICROPROP
Page 286 and 287: IN VITRO CONSERVATION AND MICROPROP
Page 288 and 289: CHAPTER 27 MICROGRAFTING OF PISTACH
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MICROPROPAGATION OF JUGLANS REGIA L
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CHAPTER 36 TISSUE CULTURE PROPAGATI
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PROPAGATION OF MONGOLIAN CHERRY AND
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410 M. PASQUAL AND E.A. FERREIRA 2.
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414 M. PASQUAL AND E.A. FERREIRA ch
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418 D.T. NHUT ET AL. its economical
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420 D.T. NHUT ET AL. Table 1. Shoot
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422 D.T. NHUT ET AL. Figure 3. Orga
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424 D.T. NHUT ET AL. mg l -1 NAA +
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426 D.T. NHUT ET AL. Nakasone, H.Y.
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428 C. LIU ET AL. C. cujete L. is c
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430 C. LIU ET AL. Table 1. Composit
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432 C. LIU ET AL. 6. Excise the sho
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434 Fresh weight per plantlet (mg)
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436 C. LIU ET AL. 4. REFERENCES Aga
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438 M. MISHRA ET AL. micropropagati
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440 M. MISHRA ET AL. each plate ino
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Section C
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446 M.G. OSTROLUCKÁ ET AL. from me
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448 M.G. OSTROLUCKÁ ET AL. regener
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450 M.G. OSTROLUCKÁ ET AL. Figure
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452 M.G. OSTROLUCKÁ ET AL. 2.6.3.
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454 counts counts M.G. OSTROLUCKÁ
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CHAPTER 42 PROTOCOL FOR MICROPROPAG
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AN medium (Anderson, 1980) MICROPRO
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MICROPROPAGATION OF VACCINIUM VITIS
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2.6. Flow Cytometry Analysis MICROP
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CHAPTER 43 MICROPROPAGATION OF BAMB
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BAMBOO MICROPROGATION BY AXILLARY S
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CHAPTER 44 IN VITRO CULTURE OF TREE
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IN VITRO CULTURE OF TREE PEONY 479
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500 M. MHATRE from various natural
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502 2.2. Disinfection of Plant Mate
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504 M. MHATRE soil in polythene bag
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506 M. MHATRE Figure 2. Pineapple,
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508 M. MHATRE Escalona, M., Lorenzo
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510 J.M. AL-KHAYRI Tunisia, Morocco
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512 J.M. AL-KHAYRI 2.1.2. Separatio
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514 2.2. Culture Medium J.M. AL-KHA
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516 J.M. AL-KHAYRI 5. Isolate callu
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518 J.M. AL-KHAYRI 3. Water the tra
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520 J.M. AL-KHAYRI expressed from c
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522 J.M. AL-KHAYRI alcohol and twic
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524 J.M. AL-KHAYRI potential. Simil
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526 J.M. AL-KHAYRI Barreveld, W.H.
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528 D.T. NHUT ET AL. arsenide chip
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530 D.T. NHUT ET AL. Figure 2. Plan
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532 D.T. NHUT ET AL. Figure 4. Plan
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534 D.T. NHUT ET AL. plantlets. The
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536 D.T. NHUT ET AL. Figure 11. Fre
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538 D.T. NHUT ET AL. Figure 13. Sub
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540 D.T. NHUT ET AL. The response o
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CHAPTER 48 IN VITRO MUTAGENESIS IN
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IN VITRO MUTAGENESIS IN BANANA 545
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3.3. Observations to be Recorded IN
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