18 Embryo Rescue
Sandra M. Reed
CHAPTER 18 CONCEPTS
• Embryo rescue procedures have been widely used for producing interspecific and
intergeneric hybrids.
• Depending on the organ cultured, embryo rescue is referred to as embryo, ovule, or
ovary culture.
• Ovule and ovary culture are more suitable than embryo culture for small-seeded species
or very young embryos.
• Cultures must be initiated before embryo abortion occurs.
• Media requirements depend on the stage of embryo development.
• Young embryos (proembryos) require a medium with a high osmotic potential.
INTRODUCTION
The term “embryo rescue” refers to a number of in vitro techniques whose purpose is to promote
the development of an immature or weak embryo into a viable plant. Embryo rescue has been
widely used for producing plants from hybridizations in which failure of endosperm to properly
develop causes embryo abortion. In embryo rescue procedures, the artificial nutrient medium serves
as a substitute for the endosperm, thereby allowing the embryo to continue its development. Embryo
rescue techniques are among the oldest and most successful in vitro procedures.
One of the primary uses of embryo rescue has been to produce interspecific and intergeneric
hybrids. While interspecific incompatibility can occur for a wide variety of reasons, one common
cause is embryo abortion. The production of small, shrunken seed following wide hybridization is
indicative of a cross in which fertilization occurred but seed development was disrupted. Embryo
rescue procedures have been very successful in overcoming this barrier to wide hybridization in a
wide range of plant materials (Collins and Grosser, 1984). In addition, embryo rescue has been
used to recover maternal haploids that have developed as a result of chromosome elimination
following interspecific hybridization.
Embryo rescue techniques also have been utilized to obtain progeny from intraspecific hybrid-
izations that do not normally produce viable seed. For example, triploids have been recovered from
crosses between diploid and tetraploid members of the same species, and progeny have been
obtained from crosses utilizing early-ripening and “seedless,” or stenospermacarpic, fruit genotypes
as maternal parents. Embryo rescue techniques have also been used in situations in which embryo
abortion is not a concern, such as for overcoming seed dormancy and studying seed development
and germination. The various applications of embryo rescue to both applied and basic plant research
0-8493-1614-6/05/$0.00+$1.50
© 2005 by CRC Press LLC 235
, 236 Plant Development and Biotechnology
have been reviewed by Bridgen (1994), Collins and Grosser (1984), Ramming (1990) and Sharma
et al. (1996).
Depending on the organ cultured, embryo rescue may be referred to as embryo, ovule, or ovary
culture. While the disinfestation and explant excision processes differ for these three techniques,
many of the factors that contribute to the successful recovery of viable plants are similar. This
chapter will begin with a discussion of general factors that should be considered when utilizing
embryo rescue and then turn to techniques specific to each type of embryo rescue procedure.
FACTORS INVOLVED IN EMBRYO RESCUE
MEDIA
Murashige and Skoog (MS) (Murashige and Skoog, 1962) and Gamborg’s B-5 (Gamborg et al.,
1968) media are the most commonly used basal media for embryo rescue studies (Bridgen, 1994).
Types and concentrations of media supplements required depend greatly on the stage of development
of the embryo.
Raghavan (1976) identified two phases of embryo development. In the heterotropic phase, the
young embryo, which is often referred to as a proembryo, is dependent on the endosperm. Embryos
initiated at this stage require a complex medium. Amino acids, particularly glutamine and aspargine,
are often added to the medium. Various vitamins may also be included. Natural extracts, such as
coconut milk and casein hydrolysate, have sometimes been used instead of specific amino acids.
Young embryos require a medium of high osmotic potential. Sucrose often serves both as a carbon
source and osmoticum. High osmotic concentration in the medium prevents precocious germination
and supports normal embryonic development. For heterotropic embryos, 232 to 352 mM (8–12%)
sucrose is commonly used. Other sugars have been successfully used instead of or in addition to
sucrose; however, sucrose has been by far the most commonly utilized sugar for embryo rescue.
The second stage of embryo development is the autotrophic phase, which usually begins in the
late heart-shaped embryo stage (Raghavan, 1976). At this time the embryo is capable of synthesizing
substances required for its growth from salts and sugar. Germination will usually occur on a simple
inorganic medium, supplemented with 58 to 88 mM (2–3%) sucrose.
Growth regulators have been extensively used in embryo rescue studies, especially for hetero-
tropic embryos; however, their effects have been highly inconsistent. In general, low concentrations
of auxins have promoted normal growth, gibberellic acid has caused embryo enlargement, and
cytokinins have inhibited growth (Sharma, 1996). In addition to supplying vitamins and amino
acids to the medium, natural extracts often also supply growth regulators.
As stated earlier, media requirements differ depending on stage of embryo development. For
cultures initiated using very young embryos, more than one media formulation may be needed.
For example, embryos of Trifolium interspecific hybrids were first cultured on a high sucrose
medium containing a moderate level of auxin and a low level of cytokinin. After 1 to 2 weeks on
this medium, embryos stopped growing. Growth resumed after they were transferred to a medium
with a lower sucrose concentration, a low level of auxin, and a moderate level of cytokinin (Collins
and Grosser, 1984).
For interspecific hybrids, it may be useful to develop media that can nurture embryos of one
or both parental species. While the nutritional needs of the hybrid may be different from the parents,
the parental media formulations will serve as a good starting point for the hybrid.
TEMPERATURE AND LIGHT
Temperature and light requirements vary among species. According to Sharma (1996), the growth
requirements of embryos often mimic those of their parents, with embryos of cool-season crops
requiring lower temperatures than those of warm-season crops. Cultures are often incubated at 25
Sandra M. Reed
CHAPTER 18 CONCEPTS
• Embryo rescue procedures have been widely used for producing interspecific and
intergeneric hybrids.
• Depending on the organ cultured, embryo rescue is referred to as embryo, ovule, or
ovary culture.
• Ovule and ovary culture are more suitable than embryo culture for small-seeded species
or very young embryos.
• Cultures must be initiated before embryo abortion occurs.
• Media requirements depend on the stage of embryo development.
• Young embryos (proembryos) require a medium with a high osmotic potential.
INTRODUCTION
The term “embryo rescue” refers to a number of in vitro techniques whose purpose is to promote
the development of an immature or weak embryo into a viable plant. Embryo rescue has been
widely used for producing plants from hybridizations in which failure of endosperm to properly
develop causes embryo abortion. In embryo rescue procedures, the artificial nutrient medium serves
as a substitute for the endosperm, thereby allowing the embryo to continue its development. Embryo
rescue techniques are among the oldest and most successful in vitro procedures.
One of the primary uses of embryo rescue has been to produce interspecific and intergeneric
hybrids. While interspecific incompatibility can occur for a wide variety of reasons, one common
cause is embryo abortion. The production of small, shrunken seed following wide hybridization is
indicative of a cross in which fertilization occurred but seed development was disrupted. Embryo
rescue procedures have been very successful in overcoming this barrier to wide hybridization in a
wide range of plant materials (Collins and Grosser, 1984). In addition, embryo rescue has been
used to recover maternal haploids that have developed as a result of chromosome elimination
following interspecific hybridization.
Embryo rescue techniques also have been utilized to obtain progeny from intraspecific hybrid-
izations that do not normally produce viable seed. For example, triploids have been recovered from
crosses between diploid and tetraploid members of the same species, and progeny have been
obtained from crosses utilizing early-ripening and “seedless,” or stenospermacarpic, fruit genotypes
as maternal parents. Embryo rescue techniques have also been used in situations in which embryo
abortion is not a concern, such as for overcoming seed dormancy and studying seed development
and germination. The various applications of embryo rescue to both applied and basic plant research
0-8493-1614-6/05/$0.00+$1.50
© 2005 by CRC Press LLC 235
, 236 Plant Development and Biotechnology
have been reviewed by Bridgen (1994), Collins and Grosser (1984), Ramming (1990) and Sharma
et al. (1996).
Depending on the organ cultured, embryo rescue may be referred to as embryo, ovule, or ovary
culture. While the disinfestation and explant excision processes differ for these three techniques,
many of the factors that contribute to the successful recovery of viable plants are similar. This
chapter will begin with a discussion of general factors that should be considered when utilizing
embryo rescue and then turn to techniques specific to each type of embryo rescue procedure.
FACTORS INVOLVED IN EMBRYO RESCUE
MEDIA
Murashige and Skoog (MS) (Murashige and Skoog, 1962) and Gamborg’s B-5 (Gamborg et al.,
1968) media are the most commonly used basal media for embryo rescue studies (Bridgen, 1994).
Types and concentrations of media supplements required depend greatly on the stage of development
of the embryo.
Raghavan (1976) identified two phases of embryo development. In the heterotropic phase, the
young embryo, which is often referred to as a proembryo, is dependent on the endosperm. Embryos
initiated at this stage require a complex medium. Amino acids, particularly glutamine and aspargine,
are often added to the medium. Various vitamins may also be included. Natural extracts, such as
coconut milk and casein hydrolysate, have sometimes been used instead of specific amino acids.
Young embryos require a medium of high osmotic potential. Sucrose often serves both as a carbon
source and osmoticum. High osmotic concentration in the medium prevents precocious germination
and supports normal embryonic development. For heterotropic embryos, 232 to 352 mM (8–12%)
sucrose is commonly used. Other sugars have been successfully used instead of or in addition to
sucrose; however, sucrose has been by far the most commonly utilized sugar for embryo rescue.
The second stage of embryo development is the autotrophic phase, which usually begins in the
late heart-shaped embryo stage (Raghavan, 1976). At this time the embryo is capable of synthesizing
substances required for its growth from salts and sugar. Germination will usually occur on a simple
inorganic medium, supplemented with 58 to 88 mM (2–3%) sucrose.
Growth regulators have been extensively used in embryo rescue studies, especially for hetero-
tropic embryos; however, their effects have been highly inconsistent. In general, low concentrations
of auxins have promoted normal growth, gibberellic acid has caused embryo enlargement, and
cytokinins have inhibited growth (Sharma, 1996). In addition to supplying vitamins and amino
acids to the medium, natural extracts often also supply growth regulators.
As stated earlier, media requirements differ depending on stage of embryo development. For
cultures initiated using very young embryos, more than one media formulation may be needed.
For example, embryos of Trifolium interspecific hybrids were first cultured on a high sucrose
medium containing a moderate level of auxin and a low level of cytokinin. After 1 to 2 weeks on
this medium, embryos stopped growing. Growth resumed after they were transferred to a medium
with a lower sucrose concentration, a low level of auxin, and a moderate level of cytokinin (Collins
and Grosser, 1984).
For interspecific hybrids, it may be useful to develop media that can nurture embryos of one
or both parental species. While the nutritional needs of the hybrid may be different from the parents,
the parental media formulations will serve as a good starting point for the hybrid.
TEMPERATURE AND LIGHT
Temperature and light requirements vary among species. According to Sharma (1996), the growth
requirements of embryos often mimic those of their parents, with embryos of cool-season crops
requiring lower temperatures than those of warm-season crops. Cultures are often incubated at 25
