Protocols for Micropropagation of Woody Trees and Fruits

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CHAPTER 16 PROTOCOL FOR DOUBLED-HAPLOID MICROPROPAGATION IN QUERCUS SUBER L. AND ASSISTED VERIFICATION B. PINTOS 1 , J.A. MANZANERA 2 AND M.A. BUENO 1 1 Lab. Forest. Biotech. CIFOR-INIA. Ctra. de la Coruña Km. 7,5. 28040 Madrid. Spain. E-mail: bueno@inia.es 2 Technical University of Madrid (UPM). ETSI Montes. 28040 Madrid. Spain. 1. INTRODUCTION Cork oak (Quercus suber L.) belongs to the family Fagaceae. The tree is robust, up to 20 metres high. The stem may reach 2 metres of diameter. The main raw material produced by this forest species is the corky bark. The cork is a demanded product of economic importance, sustaining an active cork industry. The most valuable manufacture is the cork stopper for wines of high quality. This forest species has serious drawbacks for the deployment of the classical genetic improvement programmes, mainly based on: 1) its long life and irregular reproductive cycle; 2) low correlation between traits at the juvenile and adult phases; 3) late sexual maturation; 4) the difficulty of seed conservation and of vegetative reproduction; 5) difficulty for the establishment of seed orchards; and 6) the impractical method of repeated backcrossings for the production of pure lines. Somatic embryogenesis has been used to solve the problem of rapid plant propagation of selected trees. A different approach is based on the production of pure lines through doubledhaploid plant regeneration from gametic embryos induced in anther culture. A protocol for the production of doubled-haploids of cork oak has been developed through anther embryogenesis. By this method, the microspores are subjected to a stress treatment inside the anther cultured in vitro. Those microspores leave the gametophytic pathway and react shifting their development to the sporophytic pathway by means of which haploid embryos are obtained. Later on, those embryos develop into haploid plants that can be converted into doubled-haploids. Chromosome duplication either may spontaneously occur or be induced by the application of antimitotic chemicals. 163 S.M. Jain and H. Häggman (eds.), Protocols for Micropropagation of Woody Trees and Fruits, 163–178. © 2007 Springer.
164 2.1. Induction of Gametic Embryogenesis B. PINTOS ET AL. 2. EXPERIMENTAL PROTOCOL The induction of gametic embryogenesis in Quercus suber L. was obtained from anther cultures. During the process, the following relevant factors for the embryogenic response were considered: 1. Correlation between the developmental stages of catkins, anthers and microspores. 2. Influence of a chilling pre-treatment (4° C) on the anther response. 3. Heat shock stress treatments of the anthers. 4. Effect of the addition of activated charcoal to the induction medium for gametic embryogenesis. 2.1.1. Correlation between the Developmental Stages of Catkins, Anthers and Microspores A key factor for the successful induction of embryogenesis is the adequate developmental stage of the microspore or the pollen grain. Therefore the accurate selection of anther and catkin stages containing a high proportion of embryogenic microspores is crucial. For that purpose, catkins were selected at five different phenologic stages (Figure 1A). Anthers from those stages were dissected and the respective microspores were stained with 4’-6-diamidino-2-phenylindole (DAPI) for the determination of their developmental stage (Figure 1B–E). The anthers were placed on a glass slide in a few drops of 1mg/l DAPI in PBS plus 1% Triton X-100, and tapped softly through the coverglass. Microspores were examined under a Nikon fluorescence microscope and photographed under ultraviolet light (λ=360 nm) with a digital Coolpix 4500 Nikon camera. We observed a good relationship among the phenologic stage of the cork oak catkin, the anther size and colour, and the microspore stage. 2.1.2. Catkin Selection in Quercus suber L. At the best stage for the induction of gametic embryogenesis in Quercus suber, catkins are approximately 20 mm long and 5 mm thick. Flowers of about 2 mm in size start to separate. At this stage, anthers are green-yellowish, of about 1.2 by 1.2 mm size, containing 91% of the microspores in the late uninucleated or vacuolated phase (Figure 1D). The nucleus is moved towards a pole due to the presence of a big central vacuole. These microspores are in the optimal stage for the induction of gametic embryogenesis (Pintos et al., 2005). 2.1.3. Pre-treatment on the Anther Response Branches bearing catkins (Figure 2B) were collected from selected cork oak trees, transported to the laboratory and preserved in darkness with moist cotton wrapped at the base and enveloped in aluminium foil at 4° C for one, two or three weeks. The highest embryogenic rate was obtained after a pre-treatment at 4° C during one week. Longer cold pre-treatments, e.g., two or three weeks, produce a lower frequency of embryogenesis (0.76% and 0.49% respectively).
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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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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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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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