Regeneration and transformation of Alstroemeria

C.E. van Schaik

Research output: Thesisinternal PhD, WU

Abstract

<p><em>Alstroemeria</em> or Inca Lily is an economically important cut flower in the Netherlands. The monocotyledonous ornamental <em>Alstroemeria</em> is mainly cultivated for the production of cut flowers, but there are also <em>Alstroemeria</em> pot plants and garden plants on the market. The increasing popularity for <em>Alstroemeria</em> can be attributed to its wide range of flower colours, long vase life and low energy requirement in the greenhouse. Since 1960 many efforts have been made to create elite cultivars by conventional breeding in the Netherlands. Nowadays a novel technique, called genetic modification opens the possibility to add or alter traits which cannot easily be achieved by conventional breeding.</p><p>The aim of this thesis was to develop a regeneration and transformation procedure for genetic modification of <em>Alstroemeria,</em> which could be used routinely. The most important prerequisites for genetic modification were investigated during this thesis. The first prerequisite for complete stable transformation is an efficient regeneration system. When we started this research only a very inefficient plant regeneration system was described by Gonzalez-Benito. This plant regeneration system had a regeneration frequency of 4% and used mature zygotic embryos as explants. We developed a more efficient and embryogenic regeneration system with immature zygotic embryos as explants with a regeneration frequency of 40-50% (see Chapter 2). In Chapter 3 we tried to optimise our somatic embryogenesis system and turned it into a cyclic system, thus becoming less dependent on flowering plants.</p><p>For genetic modification a regeneration system not only needs to be embryogenic and efficient, but the cells capable of regeneration need to be accessible to transformation. Histological observations revealed that the cells capable of regeneration and the transient gene expression after particle bombardment were located in cells at the surface of the callus. So regeneration and transformation took place in the same cell layers of the <em>Alstroemeria</em> callus. Another prerequisite, namely selection for transformed cells was investigated in Chapter 4. Transformed cells need to be selected between non-transformed cells, to prevent overgrow by the non-transformed cells. The herbicide PPT was found to be a good selective agent in the somatic embryogenesis system.</p><p>The two main transformation techniques nowadays are transformation by the vector <em>Agrobacterium tumefaciens</em> or direct gene transfer by the particle gun. The plant pathogenic soil bacterium <em>Agrobacterium</em> has developed a sophisticated mechanism for stably integrating part of its extrachromosomal DNA (T-DNA) into the nuclear genome of receptive plants. The particle gun is a direct gene transfer technique, small particles coated with DNA accelerate by helium pressure to penetrate the target tissue. Both transformation techniques were investigated in <em>Alstroemeria</em> (see Chapter 5).</p><p>The marker genesβ <em>-</em> glucuronidase (GUS) and luciferase (LUC) were used. The GUS assay, which is a destructive test, shows expression in the target tissue by a blue product. Later during our research we could use the non destructive LUC assay. This LUC gene from the firefly is capable of emitting light which can be detected by a very sensitive camera. By using both marker genes we optimised the two different transformation protocols. We were unable to find stably transformed callus by the <em>Agrobacterium</em> -mediated transformations. The combined transformation of the somatic embryogenesis system with particle gun bombardment revealed only chimeric transformed plants. On the other hand, the particle gun-mediated transformation of <em>Alstroemeria</em> cell suspension cultures resulted in a complete stably transformed cell suspension culture. This cell suspension had a clear positive LUC activity and PCR positive result, indicating that the culture was indeed transformed. Unfortunately, despite many attempts, we were not able to regenerate plants from this transgenic culture. Most likely, one of the reasons for failing to do so was the long time period, which this culture had to be maintained <em>in vitro</em> .</p><p>We concluded from our transformation experiment, that it is very important to know where the origin of regeneration exactly takes place. In our <em>Alstroemeria</em> somatic embryogenesis system the origin of the regenerants was from the surface of the callus, but not from single epidermal cells. Probably this is the reason why only chimeric plants were found after combined <em>Alstroemeria</em> somatic embryogenesis and particle gun bombardment transformations. The prospects for the use of embryogenic cell suspension cultures for <em>Alstroemeria</em> transformation are good. So in the near future, <em>Alstroemeria</em> transformation research has to focus on the development of embryogenic cell suspensions or a <em>de novo</em> regeneration system from single epidermal or subepidermal cells. The advantage of a cell suspension is that in a cell suspension there are many active cells (good explants for transformation) and that first a stable transformed cell suspension can be formed. The regeneration efficiency of the cell suspension is then of less importance.</p>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Jacobsen, Evert, Promotor
  • de Jeu, M.J., Promotor
Award date24 Jun 1998
Place of PublicationS.l.
Publisher
Print ISBNs9789054858904
Publication statusPublished - 1998

Fingerprint

Alstroemeria
cell suspension culture
somatic embryogenesis
luciferase
genetic engineering
cells
callus
explants
cut flowers
Agrobacterium
gene transfer
embryo (plant)
Netherlands
DNA
Lampyridae
plant pathogenic bacteria
helium
genetic markers
vase life
Lilium

Keywords

  • ornamental plants
  • amaryllidaceae
  • genetic engineering
  • recombinant dna
  • alstroemeria

Cite this

van Schaik, C. E. (1998). Regeneration and transformation of Alstroemeria. S.l.: Van Schaik.
van Schaik, C.E.. / Regeneration and transformation of Alstroemeria. S.l. : Van Schaik, 1998. 111 p.
@phdthesis{93a36fbe55c4458c8e3a40fa53eb1114,
title = "Regeneration and transformation of Alstroemeria",
abstract = "Alstroemeria or Inca Lily is an economically important cut flower in the Netherlands. The monocotyledonous ornamental Alstroemeria is mainly cultivated for the production of cut flowers, but there are also Alstroemeria pot plants and garden plants on the market. The increasing popularity for Alstroemeria can be attributed to its wide range of flower colours, long vase life and low energy requirement in the greenhouse. Since 1960 many efforts have been made to create elite cultivars by conventional breeding in the Netherlands. Nowadays a novel technique, called genetic modification opens the possibility to add or alter traits which cannot easily be achieved by conventional breeding.The aim of this thesis was to develop a regeneration and transformation procedure for genetic modification of Alstroemeria, which could be used routinely. The most important prerequisites for genetic modification were investigated during this thesis. The first prerequisite for complete stable transformation is an efficient regeneration system. When we started this research only a very inefficient plant regeneration system was described by Gonzalez-Benito. This plant regeneration system had a regeneration frequency of 4{\%} and used mature zygotic embryos as explants. We developed a more efficient and embryogenic regeneration system with immature zygotic embryos as explants with a regeneration frequency of 40-50{\%} (see Chapter 2). In Chapter 3 we tried to optimise our somatic embryogenesis system and turned it into a cyclic system, thus becoming less dependent on flowering plants.For genetic modification a regeneration system not only needs to be embryogenic and efficient, but the cells capable of regeneration need to be accessible to transformation. Histological observations revealed that the cells capable of regeneration and the transient gene expression after particle bombardment were located in cells at the surface of the callus. So regeneration and transformation took place in the same cell layers of the Alstroemeria callus. Another prerequisite, namely selection for transformed cells was investigated in Chapter 4. Transformed cells need to be selected between non-transformed cells, to prevent overgrow by the non-transformed cells. The herbicide PPT was found to be a good selective agent in the somatic embryogenesis system.The two main transformation techniques nowadays are transformation by the vector Agrobacterium tumefaciens or direct gene transfer by the particle gun. The plant pathogenic soil bacterium Agrobacterium has developed a sophisticated mechanism for stably integrating part of its extrachromosomal DNA (T-DNA) into the nuclear genome of receptive plants. The particle gun is a direct gene transfer technique, small particles coated with DNA accelerate by helium pressure to penetrate the target tissue. Both transformation techniques were investigated in Alstroemeria (see Chapter 5).The marker genesβ - glucuronidase (GUS) and luciferase (LUC) were used. The GUS assay, which is a destructive test, shows expression in the target tissue by a blue product. Later during our research we could use the non destructive LUC assay. This LUC gene from the firefly is capable of emitting light which can be detected by a very sensitive camera. By using both marker genes we optimised the two different transformation protocols. We were unable to find stably transformed callus by the Agrobacterium -mediated transformations. The combined transformation of the somatic embryogenesis system with particle gun bombardment revealed only chimeric transformed plants. On the other hand, the particle gun-mediated transformation of Alstroemeria cell suspension cultures resulted in a complete stably transformed cell suspension culture. This cell suspension had a clear positive LUC activity and PCR positive result, indicating that the culture was indeed transformed. Unfortunately, despite many attempts, we were not able to regenerate plants from this transgenic culture. Most likely, one of the reasons for failing to do so was the long time period, which this culture had to be maintained in vitro .We concluded from our transformation experiment, that it is very important to know where the origin of regeneration exactly takes place. In our Alstroemeria somatic embryogenesis system the origin of the regenerants was from the surface of the callus, but not from single epidermal cells. Probably this is the reason why only chimeric plants were found after combined Alstroemeria somatic embryogenesis and particle gun bombardment transformations. The prospects for the use of embryogenic cell suspension cultures for Alstroemeria transformation are good. So in the near future, Alstroemeria transformation research has to focus on the development of embryogenic cell suspensions or a de novo regeneration system from single epidermal or subepidermal cells. The advantage of a cell suspension is that in a cell suspension there are many active cells (good explants for transformation) and that first a stable transformed cell suspension can be formed. The regeneration efficiency of the cell suspension is then of less importance.",
keywords = "sierplanten, amaryllidaceae, genetische modificatie, recombinant dna, alstroemeria, ornamental plants, amaryllidaceae, genetic engineering, recombinant dna, alstroemeria",
author = "{van Schaik}, C.E.",
note = "WU thesis 2464 Proefschrift Wageningen",
year = "1998",
language = "English",
isbn = "9789054858904",
publisher = "Van Schaik",

}

van Schaik, CE 1998, 'Regeneration and transformation of Alstroemeria', Doctor of Philosophy, S.l..

Regeneration and transformation of Alstroemeria. / van Schaik, C.E.

S.l. : Van Schaik, 1998. 111 p.

Research output: Thesisinternal PhD, WU

TY - THES

T1 - Regeneration and transformation of Alstroemeria

AU - van Schaik, C.E.

N1 - WU thesis 2464 Proefschrift Wageningen

PY - 1998

Y1 - 1998

N2 - Alstroemeria or Inca Lily is an economically important cut flower in the Netherlands. The monocotyledonous ornamental Alstroemeria is mainly cultivated for the production of cut flowers, but there are also Alstroemeria pot plants and garden plants on the market. The increasing popularity for Alstroemeria can be attributed to its wide range of flower colours, long vase life and low energy requirement in the greenhouse. Since 1960 many efforts have been made to create elite cultivars by conventional breeding in the Netherlands. Nowadays a novel technique, called genetic modification opens the possibility to add or alter traits which cannot easily be achieved by conventional breeding.The aim of this thesis was to develop a regeneration and transformation procedure for genetic modification of Alstroemeria, which could be used routinely. The most important prerequisites for genetic modification were investigated during this thesis. The first prerequisite for complete stable transformation is an efficient regeneration system. When we started this research only a very inefficient plant regeneration system was described by Gonzalez-Benito. This plant regeneration system had a regeneration frequency of 4% and used mature zygotic embryos as explants. We developed a more efficient and embryogenic regeneration system with immature zygotic embryos as explants with a regeneration frequency of 40-50% (see Chapter 2). In Chapter 3 we tried to optimise our somatic embryogenesis system and turned it into a cyclic system, thus becoming less dependent on flowering plants.For genetic modification a regeneration system not only needs to be embryogenic and efficient, but the cells capable of regeneration need to be accessible to transformation. Histological observations revealed that the cells capable of regeneration and the transient gene expression after particle bombardment were located in cells at the surface of the callus. So regeneration and transformation took place in the same cell layers of the Alstroemeria callus. Another prerequisite, namely selection for transformed cells was investigated in Chapter 4. Transformed cells need to be selected between non-transformed cells, to prevent overgrow by the non-transformed cells. The herbicide PPT was found to be a good selective agent in the somatic embryogenesis system.The two main transformation techniques nowadays are transformation by the vector Agrobacterium tumefaciens or direct gene transfer by the particle gun. The plant pathogenic soil bacterium Agrobacterium has developed a sophisticated mechanism for stably integrating part of its extrachromosomal DNA (T-DNA) into the nuclear genome of receptive plants. The particle gun is a direct gene transfer technique, small particles coated with DNA accelerate by helium pressure to penetrate the target tissue. Both transformation techniques were investigated in Alstroemeria (see Chapter 5).The marker genesβ - glucuronidase (GUS) and luciferase (LUC) were used. The GUS assay, which is a destructive test, shows expression in the target tissue by a blue product. Later during our research we could use the non destructive LUC assay. This LUC gene from the firefly is capable of emitting light which can be detected by a very sensitive camera. By using both marker genes we optimised the two different transformation protocols. We were unable to find stably transformed callus by the Agrobacterium -mediated transformations. The combined transformation of the somatic embryogenesis system with particle gun bombardment revealed only chimeric transformed plants. On the other hand, the particle gun-mediated transformation of Alstroemeria cell suspension cultures resulted in a complete stably transformed cell suspension culture. This cell suspension had a clear positive LUC activity and PCR positive result, indicating that the culture was indeed transformed. Unfortunately, despite many attempts, we were not able to regenerate plants from this transgenic culture. Most likely, one of the reasons for failing to do so was the long time period, which this culture had to be maintained in vitro .We concluded from our transformation experiment, that it is very important to know where the origin of regeneration exactly takes place. In our Alstroemeria somatic embryogenesis system the origin of the regenerants was from the surface of the callus, but not from single epidermal cells. Probably this is the reason why only chimeric plants were found after combined Alstroemeria somatic embryogenesis and particle gun bombardment transformations. The prospects for the use of embryogenic cell suspension cultures for Alstroemeria transformation are good. So in the near future, Alstroemeria transformation research has to focus on the development of embryogenic cell suspensions or a de novo regeneration system from single epidermal or subepidermal cells. The advantage of a cell suspension is that in a cell suspension there are many active cells (good explants for transformation) and that first a stable transformed cell suspension can be formed. The regeneration efficiency of the cell suspension is then of less importance.

AB - Alstroemeria or Inca Lily is an economically important cut flower in the Netherlands. The monocotyledonous ornamental Alstroemeria is mainly cultivated for the production of cut flowers, but there are also Alstroemeria pot plants and garden plants on the market. The increasing popularity for Alstroemeria can be attributed to its wide range of flower colours, long vase life and low energy requirement in the greenhouse. Since 1960 many efforts have been made to create elite cultivars by conventional breeding in the Netherlands. Nowadays a novel technique, called genetic modification opens the possibility to add or alter traits which cannot easily be achieved by conventional breeding.The aim of this thesis was to develop a regeneration and transformation procedure for genetic modification of Alstroemeria, which could be used routinely. The most important prerequisites for genetic modification were investigated during this thesis. The first prerequisite for complete stable transformation is an efficient regeneration system. When we started this research only a very inefficient plant regeneration system was described by Gonzalez-Benito. This plant regeneration system had a regeneration frequency of 4% and used mature zygotic embryos as explants. We developed a more efficient and embryogenic regeneration system with immature zygotic embryos as explants with a regeneration frequency of 40-50% (see Chapter 2). In Chapter 3 we tried to optimise our somatic embryogenesis system and turned it into a cyclic system, thus becoming less dependent on flowering plants.For genetic modification a regeneration system not only needs to be embryogenic and efficient, but the cells capable of regeneration need to be accessible to transformation. Histological observations revealed that the cells capable of regeneration and the transient gene expression after particle bombardment were located in cells at the surface of the callus. So regeneration and transformation took place in the same cell layers of the Alstroemeria callus. Another prerequisite, namely selection for transformed cells was investigated in Chapter 4. Transformed cells need to be selected between non-transformed cells, to prevent overgrow by the non-transformed cells. The herbicide PPT was found to be a good selective agent in the somatic embryogenesis system.The two main transformation techniques nowadays are transformation by the vector Agrobacterium tumefaciens or direct gene transfer by the particle gun. The plant pathogenic soil bacterium Agrobacterium has developed a sophisticated mechanism for stably integrating part of its extrachromosomal DNA (T-DNA) into the nuclear genome of receptive plants. The particle gun is a direct gene transfer technique, small particles coated with DNA accelerate by helium pressure to penetrate the target tissue. Both transformation techniques were investigated in Alstroemeria (see Chapter 5).The marker genesβ - glucuronidase (GUS) and luciferase (LUC) were used. The GUS assay, which is a destructive test, shows expression in the target tissue by a blue product. Later during our research we could use the non destructive LUC assay. This LUC gene from the firefly is capable of emitting light which can be detected by a very sensitive camera. By using both marker genes we optimised the two different transformation protocols. We were unable to find stably transformed callus by the Agrobacterium -mediated transformations. The combined transformation of the somatic embryogenesis system with particle gun bombardment revealed only chimeric transformed plants. On the other hand, the particle gun-mediated transformation of Alstroemeria cell suspension cultures resulted in a complete stably transformed cell suspension culture. This cell suspension had a clear positive LUC activity and PCR positive result, indicating that the culture was indeed transformed. Unfortunately, despite many attempts, we were not able to regenerate plants from this transgenic culture. Most likely, one of the reasons for failing to do so was the long time period, which this culture had to be maintained in vitro .We concluded from our transformation experiment, that it is very important to know where the origin of regeneration exactly takes place. In our Alstroemeria somatic embryogenesis system the origin of the regenerants was from the surface of the callus, but not from single epidermal cells. Probably this is the reason why only chimeric plants were found after combined Alstroemeria somatic embryogenesis and particle gun bombardment transformations. The prospects for the use of embryogenic cell suspension cultures for Alstroemeria transformation are good. So in the near future, Alstroemeria transformation research has to focus on the development of embryogenic cell suspensions or a de novo regeneration system from single epidermal or subepidermal cells. The advantage of a cell suspension is that in a cell suspension there are many active cells (good explants for transformation) and that first a stable transformed cell suspension can be formed. The regeneration efficiency of the cell suspension is then of less importance.

KW - sierplanten

KW - amaryllidaceae

KW - genetische modificatie

KW - recombinant dna

KW - alstroemeria

KW - ornamental plants

KW - amaryllidaceae

KW - genetic engineering

KW - recombinant dna

KW - alstroemeria

M3 - internal PhD, WU

SN - 9789054858904

PB - Van Schaik

CY - S.l.

ER -

van Schaik CE. Regeneration and transformation of Alstroemeria. S.l.: Van Schaik, 1998. 111 p.