🧬 Ovary and embryo culture

This lesson explains the core ideas, methods, and exam-relevant applications for this topic in plant biotechnology. Focus on definitions, process steps, and practical uses for revision.

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Culture of unfertilized ovaries to obtain haploid plants from egg cell or other haploid

cells of the embryo sac is called ovary culture and this process is termed as gynogenesis.

San Noem first reported the gynogenesis in barley in 1976. Subsequently, success has

been obtained in several species including wheat, rice, maize, tobacco, sugar beet, rubber

etc. About 0.2-6% of the cultured ovaries show gynogenesis and one or two, rarely up to 8,

plantlets originate from each ovary. The rate of success varies considerably with:

 Species

 Markedly influenced by the genotype so that some cultivars do not respond at all.

e.g. In rice, japonica genotypes are far more responsive than indica genotypes.

 Stage of ovary development. In most cases, the optimum stage for ovary culture is

the nearly mature embryo sac, but in rice ovaries at free nuclear embryo sac stage

are the most responsive. Generally, culture of whole flowers, ovary and ovules

attached to placenta respond better, but in Gerbera and Sunflower isolated ovules

show better response. Cold pretreatment (24-48 hr at 4C in sunflower and 24 hr at

7C in rice) of the inflorescence before ovary culture enhances gynogenesis.

 Growth regulators:

Growth regulators are crucial in gynogenesis and at higher levels they may

induce callusing of somatic tissues and even suppress gynogenesis. Growth

regulator requirement seems to depend on species. For example, in sunflower, GR

free medium is the best, while even at low level MCPA (2-methyl-4

chlorophenoxyacetic acid) induces somatic calli and SEs. But in rice, 0.125-0.5 mg/l

MCPA is optimum for gynogenesis.

 Other Factors:

Sucrose level also appears to be critical. In sunflower, 12% sucrose leads to

gynogenic embryo production, while at lower levels somatic calli and somatic

embryos were also produced. Ovaries/ ovules are generally cultured in light, but

atleast in some species, e.g., sunflower and rice, dark incubation favours

gynogenesis and minimises somatic callusing. In rice, light may lead to degeneration

of gynogenic proembryos.

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Developmental stages

Generally, gynogenesis has two or many stages and each stage has distinct

requirements. In rice, tow stages viz ., induction and regeneration are recognized. During

induction, ovaries are floated on a liquid medium having low auxin and kept in dark, while for

regeneration they are transferred on to an agar medium with higher auxin concentration and

incubated in light.

Haploid plants generally originate from egg cell in most of the species (in vitro

parthenogenesis) but in some species, e.g., rice, they arise chiefly from synergids; in atIeast

Allium tuberosum even antipodals produce haploid plants (in vitro apogamy). As in anther

culture, gynogenesis may occur either via embryogenesis or through plantlet regeneration

from callus. In rice MCPA generally leads to a small amount of protocorm like callus

formation from which shoots and roots regenerate, while picloram promotes embryo

regeneration. In contrast, sugarbeet usually shows embryo development, while in sunflower

embryos regenerate following a callus phase. In general, regeneration from a callus phase

appears, at least for the present, to be easier than direct embryogenesis.

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Advantages

1. Gynogenetic haploids may be a valuable substitute for the production of

homozygous lines in cases where cytoplasmic male sterility prevents the use of

micropsores.

2. Reduction in the frequency of albino plants in some species especially cereals.

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Limitations

1. So far it has been successful only in less than two dozens species.

2. The frequency of responding ovaries (1-5%) and the number of plantlets/ovary (1-2)

is quite low.

3. Therefore, anther culture is preferred over ovary culture. Only in those cases where

anther culture fails, e.g. sugarbeet and for male sterile lines, ovary culture assumes

significance.

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Embryo culture

In angiosperms the embryo is the miniature sporophyte resulting from the fertilized

egg or zygote. In seed bearing plants, embryos are easily accessible as they can be

separated with relative ease from the maternal tissues and cultures in vitro under aseptic

conditions in media of known chemical composition. The culture of embryo has been

practiced by plant breeders for over half a century.

The first systematic attempt to grow the embryos of angiosperms in vitro, under

aseptic conditions was made by Hanning (1904) who cultured mature embryos of Raphanus

and the conifers Cochlearia . Subsequently, many workers raised plants by cutting embryos

excised from mature seeds. Further progress in the field of embryo culture was provided by

Liabach (1925, 1929) who demonstrated the most important practical application of this

technique. He crossed Linum perenne with Linum austriacum but obtained hybrid seeds of

very light and shriveled nature without any germinability. The excised embryos from such

seeds were cultured on moist filter paper dipped in sucrose solution. This led to the

production of hybrid plants. Since then, the technique of embryo culture has been widely

used to produce hybrids which were otherwise not possible due to embryo abortion. Further,

embryo culture method offers new refined ways to characterise the development of embryo

and related problems in plants.

The selection of plant to be used for embryo culture is normally dictated by the

problem in hand. When the goal is to obtain plants from otherwise abortive seeds, the

embryos should be excised for culture prior to the onset of abortion. Zygotic embryos, being

enclosed within the sterile environment of the ovular and ovarian tissues, do not require

surface sterilization. Entire ovules are disinfected following the standard methods of surface

sterilization and embryos are dissected out and transferred to culture medium under aseptic

conditions.

For the in vitro culture of embryo generally, it is necessary to excise them from their

surrounding tissues. The mature embryos can be isolated with relative ease by splitting

open the seeds. Seeds with a hard seed coat are dissected after soaking them in water. For

plants with minute seeds, the isolation of embryos can be done under dissecting microscope

on a sterilized slide. In plants like, orchids, where the seeds are minute and lack functional

endosperm the entire ovules having embryos are cultured on the medium.

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Types

• Culture of immature embryos originating from unripe seeds that is mainly to avoid

embryo abortion with the purpose to produce a viable plant.

• Culture of mature embryos derived from ripe seeds.

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Factors affecting the success of embryo culture

Before attempting to elucidate the application of embryo culture method, it is necessary to

analyze briefly the factors influencing the embryo culture technique.

 Genotypes

 Developmental stage of the embryo at isolation. The culture of very young embryos is

very difficult. Despite considerable progress in the field of embryo culture, embryo

rescuing seems to be difficult where embryo abortion occurs at a very early stage of

development. To culture very young embryos successfully, the embryo of a particular

species is implanted in the endosperm from another seed of the same species. For

example, in the cross of Hordeum x Secale the survival rate with the implantation

technique was 30-40 per cent as compared to one per cent with traditional method of

embryo culture. This technique is termed as embryo-nurse endosperm transplant

technique.

 Growth conditions of the mother plant

 Composition of the nutrient media

The most important aspect of the embryo culture is the selection of the right culture

medium that would support progressive and orderly development of embryos excised

at different stages of development. The requirement of culture medium depends on the

types of embryo culture. They may be either post-germinal or pre-germinal. In the case

of post-germinal embryo culture, embryos are cultured only to speed up the process

after germination. This can be achieved with less complex medium or even with

sucrose or glucose solution. In pre-germinal embryo culture, immature embryos are

cultured to get plantlets, where the embryos require a complex nutrient medium.

Refinement of nutrient medium for the culture of embryos includes modifications in the

composition of mineral salts, organic nutrients and growth regulators, as for any other

type of plant tissue cultures.

The composition of the culture medium has to be formulated in such a way to suit the

developmental phase of the embryo. There are two phases in embryo development (1)

heterotrophic phase in which the embryo draws its nutrients from the endosperm and

the surrounding maternal tissues and (2) autotrophic phase in which the embryo is

metabolically capable of synthesizing substances required for growth.

Addition of amino acids and vitamins, promoted the development of the embryo.

Casein hydrolysate, an amino acid complex has been widely used as an additive to the

embryo culture media. The natural plant extracts like coconut milk, tomato juice and

extracts of banana produce higher recovery of growth and development of embryos.

Growth hormones, especially auxins are not used in embryo culture media because of

their inhibitory role in embryo growth resulting in structural abnormalities.

 Suspensor and embryo culture

The suspensor is transitory structure found at the radicle end of the proembryo. It

promotes the growth of the young embryos and degenerate in the later stages of

embryo growth, i.e., after the formation of cotyledons. Mostly embryos cultured without

the suspension showed lesser survival and maximum necrosis thus reducing the

frequency of plantlet formation.

 Light

 Temperature

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Practical applications

Embryo rescue in wide crosses

In plants the embryo inviability occurs due to many causes, though there is normal

fertilization and development in the early stage. The impairments start subsequently,

resulting in the eventual death of embryo or from the endosperm or from the surrounding

maternal tissue.

To overcome the above barriers for obtaining the hybrids, the embryo culture

technique is effective utilized in which the nutritional relationship between the embryo and

endosperm is restored by providing the artificial medium to induce and complete growth of

hibrids embryos and is called as embryo rescuing. The demonstration of the ability of the

excised embryos from non-vialbe seeds to grow successfully in artificial medium supplied

with nutrients bypassed the problems of wide hybridization and to enable transferance of

resistant genes for pests and diseases and various environmental stresses into the

cultivated species.

The embryo culture technique is not only adopted to produce interspecific hybrids,

but aslo extended to produce viable hybrids between genera. Intergeneric hybrids have

been obtained between Hordeum and Secale ; Hordeum and Hordelymus, Triticum and

Elymus; Triticum and Secale and Tripsacum and Zea .

However, for the successful embryo rescuing in interspecific and intergeneric

crosses, the composition of the artificial nutrient medium is very important. The reason is

that the medium formulated to foster growth of embryos of one hybrids combination may not

be suitable for another. To overcome the constraints in the artificial medium in inducing the

growth of embryos, the following technique is followed in which the hybrid embryos

embedded in hybrid endosperm are removed and transplanted or implanted into the normal

endosperm. This technique is termed as embryo implantation. This tecnique was first

proposed by Pissarev and Vinogradova in 1944. The embryo implantation technique could

be an alternative to improve the crossability between two species. Kruse (1974) proposed a

similar method to rescue hybrid embryos from Hordeum x Triticale, Hordeum x Agropyron

and Hordeum x Secale crosses. The hybrid embryo is removed from a dehulled caryopsis

and placed in the correct position in the endosperm of Hordeum placed in a culture medium.

Monoploid production

The advantages of haploids as tools in genetics or plant breeding become more

apparent because of their following utility values

- they provide the quickest possible way to get homozygosity

- they may serve to recover recessives

- the gametes of monoploids remain as best source for linkage studies

- the doubled products of monoploids from crosses provide stable recombinants

- the monoploids are useful in genome homology studies

- the monoploids are ideal objects for mutation studies

- the monoploids are useful in gene transfer studies

Considering the above mentioned advantages, monoploid induction and

regeneration is considered as a powerful tool in plant breeding. The details of monoploid

production from microspores have been described in the chapter on Anther Culture. Here

how the embryo culture technique could be exploited for monoploid production is discussed.

The technique, popularly known as Bulbosum technique is exploited for producing the

monoploids and is based on making an interspecific cross with Hordeum vulgare as the

female and H. bulbosum as male. In this cross fertilization of H. vulgare by H. bulbosum

proceeds normally. During zygote development, the chromosomes of H. bulbosum are

eliminated from the cells of the developing embryo. The endosperm starts developing and

then degenerates. At this stage, the embryonic cells harbour only the set of H. vulgare

genome and show poor rate of division resulting in smaller haploid embryos. These smaller

haploid embryos with little endosperm are dissected out and cultured in vitro to produce the

haploids. Following in vitro embryo culture, the developing haploid plantlets of Hordeum

vulgare are reared and raised under normal green house conditions and chromosome

doubling is induced on established plants. This method has the advatage of throwing very

high frequencies of monoploid (haploid) induction.

Overcoming seed dormancy

The other major application of embryo culture in breeding is as a means of

overcoming seed dormancy. Seeds of certain species germinate very slowly or not at all

under normal conditions. The cause may be in the form of endogenous inhibitors, lesser

length, high temperature, storage condition and maturity of the embryo. These problems can

overcome by providing specific signals for seed germination, rightly through embryo culture.

Examples include Iris, Ilex, Viburnum, Paeonia, Brassica chinensis, Musa bulbisiana, etc .

Shortening breeding cycle of plants

Embryo culture is also useful in reducing the breeding cycle of new varieties in cases

where long dormancy causes extension of breeding cycle. Cultivated varieties of rose

generally take about a year to flower and two to three months for the formation of fruits.

Seedlings produced from cultured embryos flower in two to three months. These flowers can

serve as the male parent for further crosses, thus enabling the breeder to produce two

generations in one year or shortening the breeding cycle to three or four months. Other

example is weeping crap apple (Malusop) in which the seeds cultured in vitro produce

seedling in four months. On the other hand, seeds planted in the soil take about nine

months to germinate.

Combining embryo culture and back crossing in gene transfer

The embryo culture has been proved as a viable technique for resynthesising some

of the plant hybrids .For example Brassica napus has been resynthesised from the cross of

B. campestris/B. oleracea using embryo culture. The recent approach is back crossing the

resynthesised B. napus (2n=38) to B. campestris (2n=20), so that the genes from B.

oleracea can be transferred to B.campestris .In 1988 Quazi made an attempt in this regard

and came out with sucessful results. He got aline from the back crosses of ( B. napus/B.

oleracea )/ B. oleracea which is resistant to cabbage aphid attack. Following the same

approach Milanova and his co-workers (1991) produced cytoplasmically male sterile

tobacco plants from Nicotiana africana and N . tabaccum cross. Thus the scheme facilitates

gene transfer overcoming the species barrier.

Other applications

The embryo culture technique can be effectively engaged in seed testing of various

tree species, germinating seeds of obligate phanerogamic parasites, studying the host

pathogen relationship in seed-borne diseases and studying developmental embryogenesis.

The embryo culture technique has already established its creditability as an invaluable tool

in plant breeding and advances in embryo culture method have served to open new vistas in

the field of in vitro culture. But greater attention has to be paid to solve the minute intricacies

which remain as big hurdles in the exploitation of embryo culture.

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Embryo rescue

Distant crosses may fail due to one or more of several reasons such as inability of

pollen to germinate, failure of pollen tubes to grow or perhaps more commonly degeneration

of endosperm. When embryo fails to develop due to endosperm degeneration, embryo

culture is used to recover hybrid plants. This is called as hybrid rescue through embryo

culture. Some recent examples are the recovery of hybrids from Hordium vulgare X Secale

cereale, Triticum aestivum X Agropyron repens, H. vulgare X Triticum aestivum etc., In case

of Triticale rare combinations between Triticale and Secale develop viable seeds. But most

of the tetraploid and hexaploid wheat carry two dominant genes, Kr1 and Kr2, which prevent

seed development in crosses with Secale . The majority of the hybrid seeds is small, poorly

developed and show very poor germination. Further, seeds are obtained from only 5-10% of

the florets pollinated. The recovery of hybrid seedlings is much greater (50-70%) when

embryos from 10-14 day old caryopses are removed and cultured on a suitable medium.

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Bulbosum technique

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Principle

The fertilization proceeds readily between H. vulgare and H. bulbosum. Zygote

induction is high and chromosomes of H. bulbosum are rapidly eliminated from the cells of

developing embryo. This develops for two to five days and then aborts. In the developing

monoploid embryo cells, the division and increment is slower than the diploid cells. This

comparatively slow growth of the monoploid condition, together with the disintegration of the

endosperm leads to the formation of small embryos which have to be dissected out of the

fruits and provided with nutrients in vitro in order to complete their development. Following in

vitro embryo culture, the developing plantlets are raised under normal green house

conditions and chromosome doubling is induced on established plants.

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Advantages

• The method of hybridization followed by chromosome elimination proves to be of

general interest for haploid production in other species of Hordeum and also of

hexaploid wheat.

• It is possible to produce monoploids of barely in a cytoplasm of H. bulbosum by

using H. vulgare as male and H. bulbosum as female. Using embryo culture as

vehicle, high frequency foreign cytoplasm monoploids can be obtained.

• Hordeum species is not the only one where chromosome elimination is found in

higher plants. In Haplopoppus, monoploids have been examined with only two

chromosomes. H. bulbosum need not be the ideal partner for H. vulgare to induce

monoploids of barley via somatic chromosome elimination. There can be a range of

Hordeum that might be tried as a more efficient pattern than H. bulbosum .

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Questions

1. Embryo culture is used …………

a) To overcome embryo abortion b) To overcome seed dormancy

c) Embryo rescue in distant hybridization d) All the above

2. Ovary culture is first reported by …………

a). Northern b). San Noem c). Kano d). None of the above

3. Ovary culture is first reported in …………

a). Barley b). Sorghum c). Cotton d). None of the above

4. Embryo culture is first reported in …………

a). Raphanus b). Sorghum c). Cotton d). None of the above

5. Embryo culture is first reported by …………

a). Hanning b). San Noem c). Kano d). None of the above

6. The success of embryo culture depends on …………

a). Developmental stage of the embryo at

a). Developmental stage of the embryo at b). Growth conditions of the mother

isolation plant

c). Composition of the nutrient medium d). All the above

7. The growth hormone not used in embryo culture medium is …………

isolation

a). Auxin b). Gibberellin c). Cytokinin d). None of the above

8. The embryo imlantation technique was proposed by …………

a). Pissarev b). Vinogradova c). Both a & b d). Kruse

9. The embryo culture is used for …………

a). Embryo rescue b). Monoploid production c). To overcome seed dormancy d). All the above

10. Bulbosum technique is used for …………

a). Embryo rescue b). Monoploid production c). To overcome seed dormancy d). All the above

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Additional readings…

http://www.youtube.com/watch?v=m5JEZq0Fxuk&feature=related - video

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Summary Cheat Sheet

Quick Recall Points

• Define key terms in one line and revise their use in plant biotechnology.
• Memorize major steps, methods, and applications covered in this lesson.
• Practice exam-style distinctions between related concepts and techniques.

Exam Traps

• Do not confuse similar terms without checking context and biological level.
• Revise process order carefully; sequence-based questions are common.
• Link each method with its most likely application question.

References

1 source • [1]

[1]

Standard BSc Agriculture Plant Biotechnology notes

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