🧬 Test tube fertilization

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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The task of the plant breeder can be made difficult by any of the following eventualities:

the pollen fails to germinate on the stigma, the growth of the pollen tube in the style partially or

completely stagnates, no fertilization takes place, the fertilized egg cell does not develop in vivo

and aborts, or abscission of the ovaries occurs permanently. If no fertilization takes place after

self pollination or cross pollination then it is referred to as self incompatibility or cross

incompatibility. In some cases the plant breeder must resort to special procedures to bring

about fertilization e.g. by ovule fertilization (here the pollen is artificially brought into contact with

the ovules).

It was not until 1962 that did the idea arose of bringing about fertilization in vitro when

this was not possible in vivo . Despite the fact that little research had been carried out in this

area a few interesting examples of test tube fertilization were found.

In vitro fertilization is of particular importance if the incompatibility is present on the

stigma or in the style.

In vitro fertilization can take place in three different ways:

1. Stigma fertilization: in this method an emasculated flower is extremely sterilized and

then isolated in vitro . Pollen from a ripe anther (which has been externally sterilized) is

then placed on the stigma. This method, which is similar to fertilization in vivo, can be

used, if for example, the ovaries fall off the plant prematurely, resulting in lack of

progeny. Using stigma fertilization success has been achieved with: Nicotiana rustica, N.

tabacum, Petunia violacea, Antirrhinum majus, Pisum sativum, Lathyrus odoratus, Zea

mays and Glycine species.

2. Placental fertilization: An intact flower is externally sterilized and placenta explants

with unfertilized ovules are dissected under a stereomicroscope and inoculated onto a

nutrient medium. At the same time anthers which are still closed and at a stage where

they would be just about to open in vivo are externally sterilized. The anthers are opened

under sterile conditions and the pollen grains placed near the ovules. After this, time is

required to determine whether the pollen grains germinate, if they penetrate the embryo

sac and whether fertilization follows. Placental fertilization is practiced with members of

the Caryophyllaceae, Gossypium and Zea mays .

3. Fertilization of an isolated ovule without a placenta: This method is same as in 2

from the time that the ovule is isolated in vitro . There has been little success with this

method since it is extremely difficult to induce embryo formation in in vitro fertilized

ovules.

In vitro fertilization can be used in the following cases:

1. Placental pollination is sometimes possible when the plants are completely self

incompatible in vivo . E.g. Petunia axilaris, Petunia hybrids.

2. Cross fertilization may be possible in vitro even if it is impossible in vivo . Hybrid

plants after test tube fertilization of ovules of Nicotiana alata with pollen from

Nicotiana tabacum . Intergeneric crosses can also be achieved in vitro, as seen with

different members of the Caryophyllaceae ; for this family it has been shown that the

pollen grains germinate better with placental fertilization in vitro than on the stigma in

vivo.

3. Production of haploids by parthenogenesis.

4. The abscission of a flower or ovary is sometimes unavoidable. In such a case stigma

fertilization may be effective.

5. To study the physiology of the fertilization.

In general little is known about the conditions necessary for fertilization in vitro . However, it

seems certain that:

1. The pollen grains and the ovules must be in the correct physiological and

morphological state.

2. The choice of nutrient medium is extremely important. It is not surprising that this

choice is very difficult processes have to take place one after the other: germination

of the pollen grains, fertilization and growth of the embryo into a seed. A complex

mixture of compounds is often used to induce growth of the embryo.

3. When sterilizing flowers for use with the stigma fertilization, care should be taken that

the stigma is not in contact with the sterilizing agent for too long or the exudates on

the stigma will be dissolved.

4. With stigma fertilization it is better not to remove the sepals from the flower, since

they encourage the growth of the ovary.

5. Stigma fertilization may still be possible despite failure of placental fertilization.

6. Temperature may be a decisive factor.

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Questions

1. In vitro fertilization can take place through …………

a) Stigma fertilization b) Placental fertilization

c) Fertilization of an isolated ovule without a placenta d) All the above

2. Stigma fertilization is successful in …………

a) Nicotiana rustica b) Zea mays

c) Pisum sativum d) All the above

3. Placental fertilization is successful in …………

a) Caryophyllaceae b) Zea mays

c) Gossypium d) All the above

4. The conditions necessary for in vitro fertilization include …………

a) Physiological state of pollen and ovule b) Morphological state of pollen and ovule

c) Nutrient medium d) All the above

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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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