In vitro culture Applications

Transcription

1 In vitro culture Applications Protoplast Clonal plant propagation Virus-free plants Genetic modified plants Germoplasm bank Somatic fusion Synthetic seeds

2 What is a protoplast? The living cytoplasm of each cell, bounded by the plasma membrane, constitutes the protoplast. Removing cell walls releases large populations of spherical, osmotically fragile protoplasts (naked cells), where the plasma membrane is the only barrier between the cytoplasm and its immediate external environment.

3 Uses of protoplast for Obtaining transgenic plants Obtaining somatic hybrids gene expression, cell wall and membrane permeability studying

4 Protoplast culture steps A) Protoplast isolation B) Protoplast development C) Growth, division and plant regeneration

5 A) Protoplast isolation: Procedure Explants choice Physiological state of tissue Peeling of explants Nature and composition of enzyme Pre-plasmolysis Plasmolysis Protoplast harvesting Estimation of protoplast density Culture techniques

6 Protoplast isolation can be obtained by two procedures 1) MECHANICAL PROCEDURES involving slicing of plasmolysed tissues, are now rarely employed for protoplast isolation, but are useful with large cells and when limited (small) numbers of protoplasts are required. Recently, this approach has been used successfully to isolate protoplasts of the giant marine alga, Valonia utricularis, for patch clamp analyses of their electrical properties, including physiological changes of the plasma membrane induced by exposure of isolated protoplasts to enzymes normally used to digest cell walls(binder et al., 2003).

7 Type and Physiological state of explant. A convenient and most suitable source of protoplasts is mesophyll tissue from fully expanded leaves of young plants or new shoots Leaf tissue is popular because it allows the isolation of large number cells without killing the plamts The physiological status of the source tissue influences the release of viable protoplasts.

8 Features of leaf explants Tabacco foglia con tricomi

9 Protoplast have been isolated from a variety of tissues and organs Petioles Shoot apices Roots Fruit Coleoptiles Hypocotyls Stem Embryos Microspore Callus Cell suspension

10 Peeling off epidermid Epidermises removing is essential for allowing digestion enzyme to acts on the underneath tissues of explants cell walls How to remove epidermide: Peeling Brushing

11 Peeling

12 Brushing carburo di silicio (80-120) pennello a setole medie

13 Cell walls Parete Primaria e Secondaria:Cellulosa ed Emicelluosa Lamella mediana: Pectina

14 The release of protoplast is very much dependent on the nature and composition of enzymes Type of enzymes: Cellulase: acts on primary and secondary of cell walls. Ozonuka R10 generally used for wall degradation has been partially purified from the molds of Trichoderma ressei Pectinase : acts on middle lamella. The most frequently used is macerozyme (macerase) which was derived from the fungus Rhizopus

15 Source of enzymes Enzima Cellulasi Cellulisina Driselasi Emicellulasi Elicasi Mecerasi Pectolisai Zimoliasi Provenienza Trichoderma viride Trichoderma viride Irpex lacteus Aspergillus niger Helix pomatia Rhizopus arrhizus Aspergillus japonicus Arthrobacter luteus

16 Cell wall composition Monocotiledoni Dicotiledoni Cellulosa X X Emicellulose: Xylogucani X X Glucoarabinoxylani X Pectine X X Acidi Fenolici X Glico-Proteine X X

17 Enzymes sterilization

18 The release of protoplast is very much dependent on the nature and composition of enzymes The enzymatic isolation of protoplast can be performed in two different ways : Two-step or sequential method : pectinase first and cellulase after One step or simultaneous method

19 A) Protoplast isolation: Procedure Explants choice Physiological state of tissue Peeling of explants Nature and composition of enzyme Pre-plasmolysis Plasmolysis Protoplast harvesting Estimation of protoplast density Culture techniques

20 Pre-plasmolyses Protoplast yield and viability can be further enhanced by slicing of source (preplasmolysed) tissues, manual or enzymatic removal of the epidermis, and conditioning of donor material or its culture on media containing suitable osmotica (Davey et al., 2000a, 2004; Power et al., 2004).

21 A) Protoplast isolation: Procedure Explants choice Physiological state of tissue Peeling of explants Nature and composition of enzyme Pre-plasmolysis Plasmolysis Protoplast harvesting Estimation of protoplast density Culture techniques

22 Sterilization of substrates The high concentration of carbohydrate in the plasmolyses, suspension and culture protoplast medium do not allow to sterilize them in autoclave. Therefore, the filter sterilization by filter (0.2mm) is the best way

23 Plasmolysis Plasmolysis prior to enzymatic digestion of source tissues in salts (Frearson et al., 1973) and/or a sugar alcohol solutions, such as 13% (wt/vol) sorbitol as used for leaves of apricot (Ortin-Parraga and Burgos, 2003), reduces cytoplasmic damage and spontaneous fusion of protoplasts from adjacent cells. Addition of glycine to the enzyme mixture was essential in maximising protoplast release from cotyledons and hypocotyls of Cucumis melo and C. metuliferus, although the optimum concentration of glycine depended on the species and cultivar (Sutiojono et al., 2002). Yields from cotyledons were optimised by a 4-day dark treatment before enzyme digestion.

24 A typical plasmolyses medium composition Macro- e micro-elementi di Gamborg-B5 Mannitolo 0,5 M CaCl 2 1 gl -1 Time: 30 minute - 1 hour

25 A typical digestion medium composition Macro- e micro-elementi di Gamborg-B5 Mannitolo 0,5 M Cellulasi 15 gl -1 Macerozyme 3 gl -1 CaCl 2 1 gl -1 Hormones Digestion time: usually a citokinin 16 ore pectyolase, only2-4 ore

26 A) Protoplast isolation: Procedure Explants choice Physiological state of tissue Peeling of explants Nature and composition of enzyme Pre-plasmolysis Plasmolysis Protoplast harvesting Estimation of protoplast density Culture techniques

27 Protoplast purification The enzyme digested mixture obtained at this stage would contain, sub-cellular debris, undigested cells broken protoplasts and healthy protoplast. This mixture is purified by a combination of filtration, centrifugation and washing A. Filtration: solution containing protoplast is filtered through nylon mesh ( m) B. Centrifugation: the filtered protoplast-enzyme solution is mixed with a suitable volume of carbohydrate and centrifuge about 100Xg for 7-10 min. C. The protoplast bands is easily sucked off with a Pasteur pipette and are washed thrice and finally suspended inthe culture medium

28 Medium composition for protoplast purification Macro- e micro-elementi di Gamborg-B5 CaCl 2 1 gl-1 Carbohydrate : Saccarosio (0,5 M) or Percoll

29 Pea protoplasts in digestion medium

30 Percoll separation

31 Protoplasti di pisello (1) Protoplasti di pisello in sospensione (Percoll 0%) Protoplasti di pisello in sospensione (Percoll 10%) Protoplasti di pisello in sospensione (Percoll 15%) Protoplasti di pisello in sospensione (Percoll 30%)

32 Protoplasti di pisello (2) Protoplasti di pisello in sospensione (Percoll 40%)

33 A) Protoplast isolation: Procedure Explants choice Physiological state of tissue Peeling of explants Nature and composition of enzyme Pre-plasmolysis Plasmolysis Protoplast harvesting Estimation of protoplast density Culture techniques

34 Protoplast viability and density The most frequently used staining methods for assessing protoplast viability are: FDA Phenosafranine staining Calcofluor white The true test of protoplast viability is the ability of protoplasts to undergo continued mitotic division and regenerate plants

35 A) Protoplast isolation: Procedure Explants choice Physiological state of tissue Peeling of explants Nature and composition of enzyme Pre-plasmolysis Plasmolysis Protoplast harvesting Estimation of protoplast density Culture techniques

36 Protoplast have both maximum and minimum plating densities for growth. Published reports suggest that protoplast should be cultured at a density of 5X 10 3 to10 6 cells/ml. The concentration of protoplasts in a given preparations can be determined by the use of hemocytometer

37 Growth medium Protoplasts from different species and from different tissues of the same species may vary in their nutritional requirements. Consequently, the optimum medium for long-term culture must be determined empirically. Many media have been based on the MS (Murashige and Skoog, 1962) and B5 (Gamborg et al., 1968) formulations, with addition of an osmoticum, usually a non-metabolisable sugar alcohol, such as mannitol, or the somewhat more soluble, sorbitol.

38 Growth regulator The major growth regulators, auxins and cytokinins, are normally essential for sustained protoplast growth, although exceptions exist where only auxin is required, as carrot and A. thaliana (Dovzhenko et al., 2003). In contrast, auxins and cytokinins are detrimental to growth in citrus (Vardi et al., 1982). The growth requirements of protoplasts often change during culture, necessitating modification of medium composition, typically involving a reduction of the auxin concentration.

39 Growth regulator Phenylurea derivatives, such as N-(2- chloro-4-pyridyl)-nvphenylurea (Sasamoto et al., 2002), and brassinosteroids, which are similar structurally to animal steroidal hormones (Oh and Clouse, 1998), can promote division of protoplast-derived cell

40 Protoplast development Cell wall formation: generally starts within few hours after isolation and may take two or several days to complete the process under suitable conditions. The protoplast lose their characteristic spherical shape. Newly sinthesiesed cell wall can be demonstrated by staining with 0.1 % calcofluor white fluorescent stain.

41 Growth, division and plant regeneration A universal protocols does not exists in term of medium composition and physical parameters to maximise protoplast growth.

42 Recent examples of the application of plant protoplast