NEW SHOOT CULTURE - TISSUE CULTURE ADVANCED-2015

1. Propagation
SHOOT CULTURE : Plant Tissue Culture

2. Propagation
Cuttage
Graftage
Layerage
Division

3. Asexual Propagation
Cuttage
Graftage
Layerage
Division
Micropropagation

4. .
Welcome to the
15 Annual conference
of micropropagators
.
..
.
.

5. Definitions
Plant Tissue Culture - in vitro, aseptic plant culture
for any purpose including genetic transformation and
other plant breeding objectives, secondary product
production, pathogen elimination… or for asexual
(micropropagation) or sexual propagation

6. Definitions
Plant Tissue Culture
Micropropagation - use of tissue culture for clonal plant
propagation (including somatic embryogenesis)
In vitro seed or embryo culture - in vitro sexual
propagation

7. Characteristics of Most
Micropropagation Systems
• aseptic culture (no other organsims)
• small propagule = explant
• heterotrophic nutrition

8. History of Plant
Tissue Culture Propagation at Cornell
Lewis Knudson, 1922 - 1st in vitro embryo germination (orchid) - ie the
invention of in vitro embryo culture
Knudson, L, 1922, Nonsymbiotic germination of orchid seeds, Botanical Gazette
LXXIII: 19
Gavino Rotor, 1949 - 1st in vitro vegetative propagation.
Rotor, G, 1949, A method of vegetative propagation of Phalaenopsis species and
hybrids, AOS Bulletin (Dec 1) pp 738. - according to Arditti, (1993, Microprop of
Orchids (CH 1), Wiley) Rotor (Dr. Mac Daniels student) was 1st in vitro vegetative
propagation.
FC Stewart, 1959 - 1st somatic embryogenesis (carrot)
FC Stewart, MO Mapes, and K Mears, 1959, Growth and organized development of
cultured cells, Amer. Journal of Botany 45:653

9. Explants used for
micropropagation & embyo culture
• shoot meristem, tip, bud
• leaf or stem (internode)
• root
• anther / microspore
• ovule
• embryo (±) associated seed parts

10. Shoot Explants
Meristem tip
Actively growing shoot tip
Dormant bud

11. Forcing solution (HQC, sucrose, +/- gibberellin):
allows a dormant bud to be forced under clean (indoor)
conditions.
- Paul Read et al., 1990

12. Leaf Explants

13. Seed “explant” (propgaule)

14. Field Trip: October 18, 2001
AnTec Laboratory
Deflasking and Compotting Slipper Orchids
www.ladyslipper.com/compot2.html
Specialists in Paphiopedilum and Phramapedium (tropical ladyslipper orchids),
species and hybrids.

15. Stem Explant:
Scrophularia sp., Wansang Lim

16. Plant tissue culture systems
used for micropropagation
• shoot organ culture (axillary or nodal)
• root organ culture
• callus culture
• cell suspension culture
• somatic embryogenesis
• anther (microspore) culture

17. Plant tissue culture systems
used for micropropagation
• shoot organ culture (axillary or nodal)

18. Plant tissue culture systems
used for micropropagation
• root organ culture

19. Plant tissue culture systems
used for micropropagation
• callus culture
Http://www.cas.muohio.edu/~wilsonkg/biotech/ht
ml/greentc_.htmHttp://www.zeta.org.au/
~brianc/pcc.htm

20. Plant tissue culture systems
used for micropropagation
cell (liquid) suspension culture
Http://www.zeta.org.au/
~brianc/pcc.htm
(liquid medium,
agitation)
Cells and clumps of
cells in suspension
http://www.bio.purdue.edu/nscort/image.html

21. http://mars.cropsoil.uga.edu/homesoybean/somprot.htm
Plant tissue culture systems
used for micropropagation
somatic embryo culture

22. Plant Regeneration Pathways
• Organogenesis via organ culture
• Direct organogenesis
• Organogenesis from callus
• Somatic embryogenesis

23. Plant Regeneration Pathways
• Organogenesis via shoot organ culture
+ auxin

24. Organogenesis via shoot organ culture

25. Plant Regeneration Pathways
Organogenesis via root organ culture

26. Organogenesis via root organ culture

27. Plant Regeneration Pathways
• Direct organogenesis
African violet leaf culture
by Michael H. Renfroe
Http://www.jmu.edu/biology/biofac/facfro/cloning/cloning.html

28. Plant Regeneration Pathways
• Organogenesis from Callus:
African Violet
Use of a Protoplast Regeneration System for
African Violet Improvement
Traud Winkelmann, Institute for Breeding of
Ornamental Species Ahrensburg, Germany
http://aggie-horticulture.tamu.edu/tisscult/proto/wink/wink.html

29. Plant Regeneration Pathways
• Direct Organogenesis: Scrophularia sp., Wansang Lim

30. Plant Regeneration Pathways
• Somatic embryogenesis
http://mars.cropsoil.uga.edu/homesoybean/somprot.htm

31. http://mars.cropsoil.uga.edu/homesoybean/somprot.htm

32. Plant Regeneration Pathways
• Organogenesis via organ culture
• Direct organogenesis
• Organogenesis from callus
• Somatic embryogenesis

33. Revised to here 10/09/01

34. Micropropagation via Shoot Culture

35. Applications of Shoot Culture :
• propagation per se
• bulking plant breeding selections
• production and maintenance of disease free plants
• restoration of juvenility
• stock plant management

36. Shoot culture for propagation per se
Theoretical:
Eg. Mum
Conventional prop. -- 30K plants per year
Microprop: (based on 5 fold increase every 4 wks,
assuming unlimited labor, space, etc. and no losses):
1 > 5 > 25 > 125 > 625 > 3125 > 15,625 > 78,125
> 390,625 > 1,953,125 > 9,765,625 > 48,824,125 > 244,140,625

38. Shoot culture for propagation per se
Practical Considerations: economic niches
• French hybrid lilacs
• Raspberries
• Rhododendrons, Kalmia

39. Applications of Shoot Culture :
• propagation per se
• bulking plant breeding selections
• production and maintenance of disease free plants
• restoration of juvenility
• stock plant management

42. Applications of Shoot Culture :
• propagation per se
• bulking plant breeding selections
• production and maintenance of disease free plants
• restoration of juvenility
• stock plant management

43. Shoot culture for production
and maintenance of disease free plants
• assumption: because plant tissue cultures are
aspetic they are disease free
- contributed to the spread of orchid viruses

44. Shoot culture for production and maintenance of
bacterial and fungal disease-free plants
Culture indexing: for detection of Bacteria & fungi (not virus)
Or
X
propagate
destroy
save

45. Shoot culture for production
and maintenance of disease free plants
Virus

46. Effect of shoot tip (meristem) size on Stage I
survival and virus elimination

47. Shoot culture for elimination of virus:
examples:
• Ogalvee geraniums
• EMLA clonal apple rootstocks
• Dasheen mosaic virus of aroids
(diffenbachia, spathophyllum, syngonium)

48. Applications of Shoot Culture :
• propagation per se
• bulking plant breeding selections
• production and maintenance of disease free plants
• restoration of juvenility
• stock plant management

49. From MP Stock Plant
From Traditional
Stock Plant
% Rooting 99.5 91
Days to first root 16 20
Plant height 43.6 cm 36.1 cm
% w/ axillary bud
outgrowth 74.9 69.5
Restoration (?) of juvenility
from: Kristiansen, 1991, Post propagation growth of cuttings
from in vitro and in vivo propagated stock plants of
Ficus benjamina, Scientia Horticulturae 46: 315-322

50. Blueberry

51. Additional advantages associated with
Micropropagation via shoot culture
• Stock plant management
- Bamboo Nurseries (philodendron) - before MP 75% of gh space devoted to stock
plants
•Enhanced field performance - related to “rejuvenation”
-Red Rasp / C&W noted vigor and suckering: MP > Conventional
-thornless blackberry (Sqartz, et al, 1983)
-Strawberry - Zimmerman, 1986
-Aster, mum, and hosta (mum: 100 divisions vs. 10) - lasts 1 season (Simart in Zimmerman)
• Extend propagation season / especially combined with cold storage of shoot
and / or other types of culture
• Facilitate international export

52. Stages of Shoot Organ Culture
• Stage I - establishment
• Stage II- proliferation
• Stage III - pretransplant
• Stage IV - transplant ex vitro
Murashige, 1974

53. Objectives for Stage I
• asepsis
• survival
• stable growth

54. Briggs Nursery

55. Briggs Nursery

56. from: Sanchez & Vieitez, 1991, In vitro morphogenic potential
of basal sprouts and crown branches of mature chestnut, Tree
Physiology 8:59-70.
Effect of growth phase on stage I shoot
culture

57. Shoot Organ Culture
• Stage I - establishment
• Stage II - proliferation
• Stage III - pretransplant
• Stage IV - transplant ex vitro

58. Objectives for Stage II
• increase in number of
rootable units

59. Axillary shoot culture
Cytokinin

60. from: Sanchez & Vieitez, 1991, In vitro morphogenic potential
of basal sprouts and crown branches of mature chestnut, Tree
Physiology 8:59-70.
Effect of growth phase on stage I shoot
culture

63. Preformed vs. Adventitous Shoots
Rhododendron Montego with Tissue Proliferation

64. Shoot Organ Culture
• Stage I - establishment
• Stage II - proliferation
• (Stage III - pretransplant)
• Stage IV - transplant ex vitro

65. Objectives for Stage III
• +/- rooting
• +/- acclimitization ( light, RH)

66. Stage II – no roots

67. In vitro rooting cytokinin, auxin
Stage III

68. Shoot Organ Culture
• Stage I - establishment
• Stage II - proliferation
• Stage III - pretransplant
• Stage IV - ex vitro establishment

69. Stage IV – fog or fine mist at Briggs Nursery

70. Later stage acclimitization (Stage IV) at Briggs Nursery

71. Stage IV fog
tunnel at
KPL

72. Stage IV – Rhododendron at Prides Corner Nursery

73. Stage IV

74. Stage IV losses

75. Stage IV losses

76. Technical Considerations (How to do Microprop)
Laminar flow hood
From: Laminar Flow Hood Construction by Forester &
Berger, http://envhort.ucdavis.edu/dwb/outreach.htm

77. Technical Considerations (How to do Microprop)

78. Technical Considerations (How to do Microprop)

79. Technical Considerations (How to do Microprop)

80. Technical Considerations (How to do Microprop)

81. http://www.osmotek.com/product.htm#lifereactor
Osmotek Lifeline: Advanced products for plant tissue culture
Mechanization of Shoot Culture
*

82. http://www.osmotek.com/product.htm#lifereactor
Osmotek Lifeline: Advanced products for plant tissue culture

84. http://www.osmotek.com/product.htm#lifereactor
Osmotek Lifeline: Advanced products for plant tissue culture
Mechanization of Shoot Culture
*

85. http://www.osmotek.com/product.htm#lifereactor
Osmotek Lifeline: Advanced products for plant tissue culture

87. From TC in the home Kitchen, by Rick Walker
http://www.hpl.hp.com/botany/public_html/cp/slides/tc/tc.htm

88. From: Phytotechnology Laboratories, http://www.phytotechlab.com/
Murashige & Skoog basal nutrient medium
Technical Considerations (How to do Microprop)

89. Plant Tissue Culture Media / Additional Components
• vitamins: thiamine (vitamine B1), pyradoxine, etc.
• Phytohormnes (auxin / cytokinins)

90. Effect of auxin and cytokinin / auxin
on organogenesis in vitro
Cytokinin >
Auxin

91. Make new slide:
[Structure of cytokinin (BAP) here]
Cytokinins used in plant tissue culture
• (full spelling) BAP
• (full spelling) 2iP
• zeatin
• thiadiazuron
• kinetin

92. Thank you
Email : arputhaselvaraj@gmail.com