🧬 Factors influencing

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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Factors influencing morphogenesis

Morphogenesis in culture proceeds along a number of pathways. Of them, two are major

pathways - organogenesis and somatic embryogenesis. Organogenesis includes direct

genesis of adventitious shoots or roots and indirectly via callusing. Embryogenesis also

possesses two pathways where the outcome differs in the form "bipolar somatic

embryos" which in later stage form individual plantlets. Several factors influence the

phenomenon of morphogenesis considerably during culture. They are: genotypes,

explant, growth regulators, nutrients, other additives and physical environment.

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Genotype

In the plant kingdom, certain plant groups appeared to respond more readily in culture

than others. Members of carrot family ( Umbelliferae ) are considered to be a group that

can readily form somatic embryos in culture. However, differences in response were

observed among the different species of a genus and different cultivars in a species. It is

now well accepted that genetic factors contribute to the response of plant tissues in

culture. Though there are reports of recalcitrance among plant species to culture, this

problem can be successfully overcome by manipulation of explants, culture medium or

culture environment.

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Explant

Although all cells in a plant are considered totipotent, there are striking differences from

cell to cell and from organ to organ within a plant to regenerate plants. In general,

embryonic, meristematic and reproductive tissues appear to have greater potential for

growth and morphogenesis in culture. For woody species, it is possible to regenerate

some types of organs only when embryos or young inflorescences are cultured. The

inoculum must comprise actively dividing cells or juvenile cells. It is a well known fact

that physiological stage of the mother plant, its nutritional and environmental conditions

would also affect the explant for morphogenesis. So the mother plant should be grown in

a well controlled environment to get reproducible results even though some changes in

endogenous rhythm are not avoidable.

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Growth regulators

It is known that the control of morphogenesis in the majority of the cultures is largely a

function of the exogenous auxin/cytokinin ratio. High concentrations of kinetin cause

shoot initiation, whereas high levels of auxin favour rooting. In somatic embryogenesis,

auxin is required for induction of embryonic cells and maintenance of proliferative

growth. Embryo formation can be induced by transferring the callus to less auxin

medium or a medium lacking auxin. Plant growth regulators other than auxins and

cytokinins have been shown to play an important role in the induction and control of

morphogenesis. Gibberellic acid has been used most successfully to obtain rapid growth

of shoot apices and somatic embryos into plants.

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Nutrient medium

Components of nutrient medium play critical roles in controlling morphogenesis in

culture. Effects of many inorganic and organic nutrients have been studied extensively.

One of the most important components of the medium in effecting morphogenesis is the

source and concentration of nitrogen. Supply of high levels of reduced nitrogen appears

suitable to shoot formation and essential to somatic embryogenesis. This is supplied in

the form of ammonium nitrate and sometimes substituted with amino acids such as

glutamine, glycine and alanine and their amides. Presence of potassium in the medium

enhances embryogenesis.

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Other additives

Supplementation of medium with casein hydrolysate and coconut milk also favour the

morphogenesis in vitro . Coconut milk has been employed extensively as a medium

component for somatic embryogenesis.

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Culture environment

Temperature, photoperiod, light intensity and osmotic concentration are other factors

that may have determining role in organogenesis and embryogenesis. The optimum

temperature for culture is 24 ± 2 [o] C. Low temperature treatment of explants prior to

culture favours their regenerative ability. Light also exerts a strong morphogenetic effect

on plants in culture. Usually cultures produce shoots but the period of lighting should be

maintained according to the photoperiodism of normal environment. The blue region of

the spectrum promotes shoot formation and red light favours rooting. In the light, the

somatic embryos of carrot formed plants; in the absence of light etiolation occurred.

Overall osmotic concentration of a medium can also exert a profound effect on

morphogenesis. Increased osmotic levels in medium enhance shoot and somatic

embryo formation. The osmotic level can be increased by adding additional sucrose.

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Loss of morphogenetic ability

Cultures in vitro capable of morphogenetic potential initially lose the ability if they are

subcultured repeatedly. Such subcultures may bring the changes at genetic, epigenetic

and physiological levels. Variation in ploidy level of cells cultured is the usual change

occurring at genetical level. Such variations may be either polyploidy or aneuoploidy.

Sometimes gene mutations also occur in the cultured cells.

The epigenetic level changes occurring in culture are partially stable but reversible.

Habituation to a partial particular component may produce morphogenetic loss in in vitro

culture. For example, the embryogenic cultures grown in auxin plus medium would

produce somatic embryos when the cultures are transferred to auxin free medium. The

continuous culturing of callus or suspensions would lose the morphogenetic potential.

This may be due to higher concentration of endogenous auxin. But these cultures can be

made to produce embryos by depleting endogenous auxin level. For this the medium

should have activated charcoal which has the potential to absorb certain amount of

auxin.

Reduced growth rate less friability and senescence of cultures are the changes that

occur at physiological level. These changes are temporary and unstable. By providing

optimum chemical and physical environment, such morphogenetic losses can be

overcome. Thus there are many reasons for the loss of morphogenetic ability by

cultures, but there are indications of number of techniques that will help to reduce, if not

eliminate, the problem.

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Culture vessel to soil

The cellular totipotency is exploited in basic and applied aspects of plant science. This

potential is not blocked with mere demonstration of organogenesis or somatic

embryogenesis, but effectively utilized in propagating and producing entire plantlets,

similar to mother plant and new genotypes respectively. The success of this technique

depends on the method followed to establish plantlets in the soil, which have been

cultured in an entirely new environment. The method requires details on rate of

multiplication of a particular explant and the rate of establishment of regenerated

plantlets in soil. Adequate knowledge on manipulation of media, explant and culture

environment to maintain the rate of multiplication at maximum is available. Having

obtained a large number of regenerated plantlets, it is customary and necessary to

transfer them to natural conditions. This is a critical period since the plantlets removed

from the controlled environment of test tube or flask is going to face the real world.

Under in vitro conditions, the plantlets have a carefully controlled supply of nutrient,

humidity, temperature and photoperiod. The high humidity prevailing under culture

conditions induces rapid shoot growth and proliferation. During this time, cuticle

coverings of leaves and root hairs are poorly developed. If such plants are transferred to

natural conditions, there would be substantial loss of water and desiccation due to

cuticular and stomatal transpiration. So care must be taken during transfer of plantlets

from in vitro condition to natural condition. Important points to be considered during

transfer of plantlets to soil are:

 Plantlets should be allowed to develop a good root system. The cultures with shoots

may be transferred to a medium containing a weaker auxin for the better rooting.

 If the plantlets have been grown on agar-solidified medium, the agar may be

removed by gentle washing with warm water.

 Damage to the root system should be avoided.

 After washing, the plantlets may be kept under higher intensity of light than the

intensity of culture room for five to six days.

 The plantlets are then carefully planted in small plastic cups and the young roots

surrounded with fine sand. It is better to sterilize the peat soil mixture in an

autoclave to eliminate microbial pathogens.

 The small potted plantlets should be transferred to a controlled environment

chamber, where control of light, temperature and humidity are possible.

 Then plantlets may be kept in mist chamber for increasing periods of light and

temperature. During this hardening period, the plants will develop normal cuticular

system with good rooting.

The above mentioned steps make regenerants to grow under natural conditions is

collectively called as hardening and this process enhances the plant survival after

transplanting.

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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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