Protocols for Micropropagation of Woody Trees and Fruits

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2.6. Flow Cytometry Analysis MICROPROPAGATION OF VACCINIUM VITIS-IDAEA L. 463 Flow cytometry analysis was perfomed to determine potential undesirable variations in genome size induced during in vitro regeneration process. Three clones of V. vitis-idaea cv. ‘Red Pearl’ derived by adventitious regeneration and one meristemderived clone were analysed. Flow cytometry analyses were performed on Ploidy analyzer PA II, with mercury arc lamp using UV excitation. The samples were prepared from young leaves of in vitro plants of Vaccinium vitis-idaea L. by two step procedure using reagent kit Partec CyStain UV precise P (Partec GmbH, Münster, Germany) containing Extraction Buffer and Staining Buffer with DAPI: – Approximately 20 mg of young leaf tissue were chopped with a sharp razor blade in a plastic Petri dish containing 400 µl Extraction Buffer for 30–60 s. After incubation in Extraction Buffer for 1–2 min at the room temperature the sample was filtered through a 50 µm filter. – Staining Solution 1.6 ml was added to the test tube and sample was incubated for 30–60 s. Samples were analysed in flow cytometer in the blue fluorescence channel. The histograms obtained after nuclear DNA analysis contained a single dominant peak corresponding to nuclei in G1 phase of the cell cycle with 2C DNA content. Very few nuclei were found having 4C DNA content. The distribution of nuclear DNA content obtained after the analysis of nuclei isolated from meristem-derived clone was similar to those of adventitious regeneration-derived clones (Figure 3A–D). It can be stated that all tested clones are characterized by significant genetic stability and polyploid cells occurred in tissues individually without a significant tendency towards polyploidization. These finding suggest that no changes in ploidy levels occurred during regeneration process (Gajdošová et al., 2006). Considering the results of flow cytometry analyses, it seems highly probable that the stability of ploidy level under in vitro conditions is a more general phenomenon in Vaccinium spp. plants. 3. CONCLUSION In conclusion we can state, that an efficient cloning protocol was developed for V. vitis-idaea L. cultivars, which enables large-scale propagation of high quality, true to type plants for need of practice. Our results, as well as results of other authors, prevailingly confirmed that in vitro regeneration ability is highly genotype depending, therefore small protocol modifications may be necessary in different cultivars. Acknowledgements. The work was supported by Slovak Grant Agency VEGA, project no. 2/5128/25 and COST 863 Action.
464 counts counts 800 640 480 320 180 400 320 240 160 PK1 PK2 A. GAJDOŠOVÁ ET AL. 0 A 0 C 0 200 400 FL4 600 0 200 400 FL4 600 80 PK1 PK2 Figure 3. Flow cytometry histograms of lingonberry cv.‘Red Pearl’ A – meristem-derived clone, B–D – adventitious regeneration-derived clones. 4. REFERENCES Anderson, W.C. (1980) Tissue culture propagation of red and black raspberries, Rubus idaeus and R. occidentalus. Acta Hort. 112, 124–132. Finn, Ch.E., Luby, J.J., Rosen, C.J. & Ascher, P.D. (1991) Evaluation in vitro of blueberry germplasm for higher pH tolerance. Journal of Amer. Soc. Hort. Sci., 116, 312–316. Gajdošová, A., Ostrolucká, M.G., Libiaková, G., Ondrušková, E. & Šimala, D. (2006) Microclonal propagation of Vaccinium sp. and Rubus sp. and detection of genetic variability in culture in vitro. Journal of Fruit and Ornamental Plant Research, 14, 61–76. Gustavsson, B.A. & Trajkovski, V. (1999) ‘Ida’ and ‘Linnea’– Novel lingonberry cultivars with commercial potential. Fruit Varieties Journal 4, 228–231. Ostrolucká, M.G., Gajdošová, A. & Libiaková, G. (2002) Influence of zeatin on microclonal propagation of Vaccinium corymbosum L. Propagation of Ornamental Plants 2, 14–18. Ostrolucká, M.G., Libiaková, G., Ondrušková, E. & Gajdošová, A. (2004) In vitro propagation of Vaccinium species. Acta Universitatis Latviensis Biology 676, 207–212. Ondrušková, E., Ostrolucká, M.G. & Hraška, Š. (2006) Influence of zeatin and 2iP on in vitro propagation of Vaccinium vitis-idaea L. Propagation of Ornamental Plants 6, 194–200. counts counts PK1 PK2 PK1 PK2 0 B 0 D 0 200 400 FL4 600 0 200 400 FL4 600 400 320 240 160 80 400 320 240 160 80
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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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CHAPTER 27 MICROGRAFTING OF PISTACH
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MICROGRAFTING OF PISTACHIO Constitu
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MICROGRAFTING OF PISTACHIO 2.2.2. C
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MICROGRAFTING OF PISTACHIO 6. Incub
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MICROGRAFTING OF PISTACHIO 9. The r
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CHAPTER 28 PROTOCOL FOR MICROPROPAG
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MICROPROPAGATION OF CASTANEA SATIVA
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CHAPTER 29 MICROPROPAGATION OF CASH
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MICROPROPAGATION OF CASHEW 2.2.1. E
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MICROPROPAGATION OF CASHEW axillary
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MICROPROPAGATION OF CASHEW Table 1.
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MICROPROPAGATION OF CASHEW shade an
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CHAPTER 30 IN VITRO MUTAGENESIS AND
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IN VITRO MUTAGENESIS AND MUTANT MUL
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336 R.I. IYER compounds (Iyer et al
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A 338 2.2. Culture Medium R.I. IYER
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340 R.I. IYER Figure 2. Formation o
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342 R.I. IYER Figure 3. Formation o
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344 R.I. IYER Rao, Y.S., Mathew, K.
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346 B.K. BISWAS AND S.C. GUPTA marg
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348 B.K. BISWAS AND S.C. GUPTA (iv)
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350 B.K. BISWAS AND S.C. GUPTA 2.4.
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352 B.K. BISWAS AND S.C. GUPTA Figu
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354 B.K. BISWAS AND S.C. GUPTA tree
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356 B.K. BISWAS AND S.C. GUPTA Figu
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358 B.K. BISWAS AND S.C. GUPTA Drew
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CHAPTER 33 MICROPROPAGATION PROTOCO
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MICROPROPAGATION FOR MICROSPORE EMB
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374 L. TIAN AND S.I. SIBBALD younge
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376 L. TIAN AND S.I. SIBBALD Table
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378 L. TIAN AND S.I. SIBBALD 2.4. R
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CHAPTER 35 MICROPROPAGATION OF JUGL
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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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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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