This document discusses various techniques in plant biotechnology including embryo rescue, somaclonal variation, and cryopreservation. Embryo rescue involves culturing immature or weak embryos on artificial nutrient media to allow their development. Somaclonal variation refers to genetic and phenotypic changes that can occur in plants regenerated from tissue culture. Cryopreservation aims to preserve plant cells and tissues in a frozen state at ultra-low temperatures like liquid nitrogen. The key steps involve adding cryoprotectants, freezing, storage, thawing, and regeneration of plants. These techniques have various applications for breeding programs and conservation of plant genetic resources.

1. Embryo rescue,
Somaclonal Variation,
Cryopreservation
By,
Abhinava J V
BIRAC Innovation Fellow
University of Agricultural Sciences, Dharwad

2. Embryo rescue

3. • 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 also have been utilized to obtain progeny from
intraspecific hybridizations that do not normally produce viable seed.

4. Factors involved in embryo rescue
• Murashige and Skoog (MS) (Murashige and Skoog, 1962) and Gamborg’s
B-5 media are the most commonly used basal media for embryo rescue
studies.
• Types and concentrations of media supplements required depend greatly on
the stage of development of the embryo.
• Embryos initiated at heterotropic phase stage(proembryo) 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.
• Sucrose often serves both as a carbon source and osmoticum
[232 to 352 mM (8–12%) ].
Media

5. • In the autotrophic phase, which usually begins in the late heart-shaped
embryo stage. 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.
• In general, low concentrations of auxins have promoted normal growth,
gibberellic acid has caused embryo enlargement, and cytokinins have
inhibited growth. In addition to supplying vitamins and amino acids to the
medium, natural extracts often also supply growth regulators.
• Media requirements differ depending on stage of embryo development. For
cultures initiated using very young embryos, more than one media
formulation may be needed.

6. Temperature and light
• 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 usually initially cultured in the dark to prevent precocious
germination, but are moved to a lighted environment to allow chlorophyll
development after 1 to 2 weeks in the dark.
Time of culture
• Cultures are often initiated at various intervals following pollination to
maximize chances of recovering viable plants. Since an interaction between
media and time of culture is expected, it is important to test a range of
media ranging from complex with high sucrose to simple with low sucrose
at the various culture times.

7. General embryo rescue procedures
• It is the most commonly used embryo rescue procedure is embryo culture, in
which embryos are excised and placed directly onto culture medium.
• Fruit from controlled pollination of greenhouse- or field grown plants is
collected prior to the time at which embryo abortion is thought to occur. Since
embryos are located in a sterile environment, disinfestation of the embryo itself
is not required. In some cases, the entire ovary is surface-sterilized.
• The embryo will be placed directly into culture after its excision so that it does
not become dry. For heart-shaped and younger embryos, the embryo should be
excised with the suspensor intact.
Embryo culture

8. Ovule culture
• Embryos are difficult to excise when very young or from small-seeded
species. To prevent damaging embryos during the excision process, they are
sometimes cultured while still inside the ovule. This technique is referred to
as ovule culture or in ovolo embryo culture.
• As with embryo culture, ovaries are collected prior to the time at which
embryo abortion is thought to occur. The ovary is surface sterilized and the
ovules removed and placed into culture.
• This step ranges from extremely easy to accomplish, for large-seeded species
in which only a single ovule is present, to time-consuming and difficult, for
small-seeded polyovulate species.

9. Ovary culture
• In ovary or pod culture, the entire ovary is placed into culture. Ovaries
are collected and any remaining flower parts removed. Disinfestation
protocols must remove surface contaminants without damaging the
ovary. The ovary is placed into culture so that the cut end of the
pedicel is in the medium. At the end of the experiment, seed are
removed from the fruit that develop in culture.

11. Application of Embryo Rescue
• Overcoming dormancy- in some fruit plants embryos require period of
after ripening before germination. for example Prunus, Taxus etc.
• Breeding cycle shortening- seeds witch takes 10-20 days for maturation
places on in vitro culture to overcome maturation time.
• Inter specific hybrids recovery- some time distant crossed may fail cause
of inability pollen to germinate, failure of pollen tube to grow, degeneration
of endo-sperm, all causes fail to embryo germination so this problem over
come by embryo rescue.

12. Somaclonal Variation

13. • Somaclonal variation is defined as genetic and phenotypic variation
among clonally propagated plants from a single donor clone.
• Somaclonal variation caused by the process of tissue culture is also
called tissue culture induced variation to more specifically define the
inducing environment. Somaclonal variation can be manifested as
either somatically or meiotically stable events.
• Somatically stable variation includes phenotypes such as habituation
of cultures and physiologically induced variation observed among
primary regenerates.
• Meiotically heritable variation occurs and is most important in
situations where the end product, the tissue culture is propagated and
sold as seed.

14. Application of Somaclonal Variation
• Somaclonal variation is one of the useful source of introducing genetic
variations that could be of value to plant breeders.
• Single gene mutation in nuclear or organelle genome may give the best
available variety in vitro that has a specific character.
• Various cell lines selected in vitro may prove potentially applicable to
agriculture and industry like resistance to herbicide, pathotoxin, salt or
aluminium.
• Variability in cell cultures has played a useful role in synthesis of
secondary metabolites on a commercial scale.

15. Somaclonal variants in crop plants
Rice:
• Significant improvements relative to parent were observed for seed weight,
seed proteins percentage, tiller number, panicle length and time of flowering.
Wheat:
• Variations were manifested for gliadin proteins in seed, grain colour, plant
height, heading date and yield.
Maize:
• Plants regenerated from selected cell lines were resistant both to T-toxin and
to infection to Drechslera maydis causing southern leaf blight.

16. Potato:
• Somaclonal variants were selected for resistance to Phytopthora infestans
and to its multiple races and resistance to early blight.
Tomato:
• Somaclones were isolated with variant phenotypes, such as recessive
mutation for male sterility, resistance to Fusarium oxysporium, jointless
pedicel , tangerine virescent leaf, flower and fruit colour.
Sugarcane:
• Somaclonal variants have been isolated by different workers for cane yield,
sugar yield and resistance to smut disease caused by Ustilago scitamini,
downey mildew caused by Helminthosporium sacchari.

17. Cryopreservation

18. • Cryopreservation (Greek, krayos-frost) literally means preservation in
the frozen state.
• The principle involved in cryopreservation is to bring the plant cell and
tissue cultures to a zero metabolism or non-dividing state by reducing the
temperature in the presence of cryoprotectants.
• It was done by using,
• Over solid carbon dioxide (at -79°C)
• Low temperature deep freezers (at -80°C)
• In vapour phase nitrogen (at -150°C)
• In liquid nitrogen (at -196°C)
Among these, the most commonly used cryopreservation is by employing
liquid nitrogen. At the temperature of liquid nitrogen (-196°C), the cells
stay in a completely inactive state and thus can be conserved for long
periods.

19. Mechanism of Cryopreservation:
• The technique of freeze preservation is based on the transfer of water
present in the cells from a liquid to a solid state.
• Due to the presence of salts and organic molecules in the cells, the cell
water requires much more lower temperature to freeze (even up to -
68°C) compared to the freezing point of pure water (around 0°C).
• When stored at low temperature, the metabolic processes and
biological deteriorations in the cells/tissues almost come to a standstill.

20. Precautions/Limitations for Cryopreservation:
• Formation ice crystals inside the cells should be prevented as they
cause injury to the organelles and the cell.
• High intracellular concentration of solutes may also damage cells.
• Sometimes, certain solutes from the cell may leak out during freezing.
• Cryoprotectants also affect the viability of cells.
• The physiological status of the plant material is also important.

21. Process of Cryopreservation:
1. Development of sterile tissue cultures
2. Addition of cryoprotectants and pretreatment
3. Freezing
4. Storage
5. Thawing
6. Re-culture
7. Measurement of survival/viability
8. Plant regeneration.

23. Development of sterile tissue culture:
• The selection of plant species and the tissues with particular reference
to the morphological and physiological characters largely influence the
ability of the explant to survive in cryopreservation.
• Any tissue from a plant can be used for cryopreservation e.g.
meristems, embryos, endosperms, ovules, seeds, cultured plant cells,
protoplasts, calluses. Among these, meristematic cells and suspension
cell cultures, in the late lag phase or log phase are most suitable.

24. Addition of cryoprotectants and pretreatment:
• Cryoprotectants are the compounds that can prevent the damage caused to
cells by freezing or thawing.
• The freezing point and super-cooling point of water are reduced by the
presence of cryoprotectants. As a result, the ice crystal formation is retarded
during the process of cryopreservation.
• There are several cryoprotectants which include dimethyl sulfoxide
(DMSO), glycerol, ethylene, propylene, sucrose, mannose, glucose,
proline and acetamide.
• Among these, DMSO, sucrose and glycerol are most widely used.
• Generally, a mixture of cryoprotectants instead of a single one is used for
more effective cryopreservation without damage to cells/tissues.

25. Freezing:
Four different types of freezing methods are used:
1. Slow-freezing method:
• The tissue or the requisite plant material is slowly frozen at a slow cooling
rates of 0.5-5°C/min from 0°C to -100°C, and then transferred to liquid
nitrogen.
2. Rapid freezing method:
• This technique is quite simple and involves plunging of the vial containing
plant material into liquid nitrogen. During rapid freezing, a decrease in
temperature -300° to -1000°C/min occurs. The freezing process is carried out
so quickly that small ice crystals are formed within the cells.

26. 3. Stepwise freezing method:
• This is a combination of slow and rapid freezing procedures (with the
advantages of both), and is carried out in a stepwise manner. The plant
material is first cooled to an intermediate temperature and maintained there
for about 30 minutes and then rapidly cooled by plunging it into liquid
nitrogen. Stepwise freezing method has been successfully used for
cryopreservation of suspension cultures, shoot apices and buds.
4. Dry freezing method:
• Some workers have reported that the non-germinated dry seeds can survive
freezing at very low temperature in contrast to water-imbibing seeds which
are susceptible to cryogenic injuries. In a similar fashion, dehydrated cells
are found to have a better survival rate after cryopreservation.

27. Storage:
• Maintenance of the frozen cultures at the specific temperature is as
important as freezing. In general, the frozen cells/tissues are kept for
storage at temperatures in the range of -70 to -196°C.
• However, with temperatures above -130°C, ice crystal growth may
occur inside the cells which reduces viability of cells. Storage is
ideally done in liquid nitrogen refrigerator — at 1 50°C in the vapour
phase, or at -196°C in the liquid phase.

28. Thawing:
• Thawing is usually carried out by plunging the frozen samples in ampoules
into a warm water (temperature 37-45°C) bath with vigorous swirling.
• By this approach, rapid thawing (at the rate of 500- 750°C min-1) occurs, and
this protects the cells from the damaging effects ice crystal formation.
• As the thawing occurs (ice completely melts) the ampoules are quickly
transferred to a water bath at temperature 20-25°C.
• This transfer is necessary since the cells get damaged if left for long in warm
(37-45°C) water bath.
• For the cryopreserved material (cells/tissues) where the water content has
been reduced to an optimal level before freezing, the process of thowing
becomes less critical.

29. Re-culture:
• In general, thawed germplasm is washed several times to remove
cryoprotectants.
• This material is then re-cultured in a fresh medium following standard
procedures.
• Some workers prefer to directly culture the thawed material without
washing. This is because certain vital substances, released from the
cells during freezing, are believed to promote in vitro cultures.

30. Measurement of survival/viability:
• The techniques employed to determine viability of cryopreserved cells are
the same as used for cell cultures .Staining techniques using triphenyl
tetrazolium chloride (TTC), Evan’s blue and fluorescein diacetate (FDA)
are commonly used.
• The best indicator to measure the viability of cryopreserved cells is their
entry into cell division and regrowth in culture. This can be evaluated by
the following expression.

31. Plant regeneration:
• The ultimate purpose of cryopreservation of germplasm is to
regenerate the desired plant.
• For appropriate plant growth and regeneration, the cryopreserved
cells/tissues have to be carefully nursed, and grown.
• Addition of certain growth promoting substances, besides maintenance
of appropriate environmental conditions is often necessary for
successful plant regeneration.

32. Applications of Cryopreservation:
• Maintenance of stock cultures: Plant materials of several species can be
cryopreserved and maintained for several years, and used as and when needed.
• Cryopreservation is an ideal method for long term conservation of cell cultures
which produce secondary metabolites (e.g. medicines).
• Disease (pathogen)-free plant materials can be frozen, and propagated whenever
required.
• Recalcitrant seeds can be maintained for long.
• Conservation of somaclonal and gametoclonal variations in cultures.
• Plant materials from endangered species can be conserved.
• Conservation of pollen for enhancing longevity.
• Rare germplasms developed through somatic hybridization and other genetic
manipulations can be stored.