INVITRO CULTURE: TECHNIQUES, APPLICATIOSNS & ACHIEVEMENTS. INVITRO TECHNIQUES AND BIOTECHNOLOGY USE IN AGRICULTURE AND CROP IMPROVEMENT. APPLICATIONS OF VARIOUS BIOTECHNOLOGICAL TECHNIQUES AND METHODS. TISSUE CULTURE, MICROPROPAGATION, EMBRYO CULTURE, ANTHER CULTURE, POLLEN CULTURE, ENDOSPERM CULTURE, OVULE CULTURE, OVARY CULTURE, ETC.

1. IN-VITRO CULTURE : TECHNIQUES,
APPLICATIONS & ACHIEVEMENTS
BY: AYUSH MISHRA

2. IN VITRO CULTURE
•In-Vitro is a Latin word meaning in
glass. It refers to any process carried
out in sterile culture under controlled
conditions in the laboratory.
•The technique or process of
maintaining or cultivating cells or
tissues derived from living organisms
in culture medium is called In Vitro
Culture.

3. GENERAL REQUIREMENTS
FOR IN VITRO CULTURE
1) LABORATORY ORGANISATION
• Media room for preparation, sterilization and storage.
• Facilities for washing lab wares and explants.
• Culture rooms or incubators for maintaining proper temperature,
humidity and light requirement.

4. BIOTECHNOLOGY LAB

5. 2) CULTURE MEDIA
•The formulation or the medium on
which the explant is cultured is called
culture medium. It is composed of
various nutrients required for proper
culturing.
•Different types of plants and organs
need different composition of culture
media.
•E.g. MS medium, LS medium, B5
medium, White's medium

6. 3) ASEPTIC CONDITIONS
•Maintenance of aseptic conditions is
most critical and difficult aspect of in-
vitro culturing experiments.
•Aseptic condition mean the conditions
free from any type of microorganisms.
•Fungi and Bacteria are most common
contaminants.
•Sterilization is done to maintain
aseptic conditions.

7. Basic Steps For In Vitro Culturing of
Plants
1) Selection and Sterilization of explant
•Suitable explant is selected and is then
excised from the donor plant. Explant is then
sterilized using disinfectants.
2) Preparation and Sterilization of Culture
Medium
•A suitable culture medium is prepared with
special attention towards the objectives of
culture and type of explant to be cultured.
•Prepared culture medium is transferred into
sterilized vessels and then sterilized in
autoclave.

8. 3) Inoculation
•Sterilized explant is inoculated
(transferred) on the culture medium
under strict aseptic conditions.
4) Incubation
•Cultures are then incubated in the
culture room where appropriate
conditions of light, temperature and
humidity are provided for successful
culturing.

9. 5) Sub Culturing
• Cultured cells are transferred to a
fresh nutrient medium to obtain the
plantlets.
6) Transfer of Plantlets
• After the hardening process (i.e.
acclimatization of plantlets to the
environment), the plantlets are
transferred to green house or in pots.

11. ADVANTAGES OF IN
VITRO CULTURE
1) It permit a species specific,
simpler, more convenient & more
detailed analyses.
2) It simplifies the system under
study, so the investigator can focus
on small no. of components.
3) It allows us to study human cells
without extrapolation, from an
experimental animal cell or tissue.

12. 4) In Vitro methods can be miniatured
automated yielding high throughput
screening method for testing
molecules in pharmacology or
toxicology.
5) It is not time bound & season bound.

13. DIFFERENT TECHNIQUES OF
IN VITRO CULTURES
1) MICROPROPAGATION
2) EMBRYO CULTURE
3) OVULE CULTURE
4) OVARY CULTURE
5) ANTHER CULTURE
6) POLLEN CULTURE
7) ENDOSPERM CULTURE

14. MICROPROPAGATION
• It is the practice of rapidly multiplying stock plant material to produce
large number of Progeny.
• Frederic Campion Steward discovered micro propagation and plant
tissue culture in late 1950's.
• Department of Biotechnology, GOI constituted CMRTD which looks for
the plant Species where Micro Propagation can be done.

16. Steps Involved in
MICROPROPAGATION
1) Selection and Propagation of Mother
plant.
•sterilisation of mother plant takes place
2) Initiation of Culture
•explant placed into growth media
3) Multiplication
•explant transferred to shoot media, shoots
can be constantly divided
4) Rooting
•explant transferred to root media
5) Transferred to Soil
•explant returned to soil, hardened off

17. APPLICATION OF
MICROPROPAGATION
1) High rate of plant propagation
• A large no. of plant can be grown from a piece of
plant tissue within a short period. It can also be
carried throughout the year.
2) Production of disease free plant
•It is used to successfully produce disease free
plants in sweet potato, cassava and yam
3) Production of seeds in some Crops
•This is required in certain plant where limitation
for seed production is high degree of genetic
conservation. E.g. Cauliflower, Onion.

18. 4) Cost- Effective Process
• It requires minimum growing space, Thus millions of plant species
can be maintained inside culture in a small room or nursery.
5) Automated Micro Propagation
• Bioreactors have been setup at large scale for multiplication of
shoots and bulbs. Robots are used instead of manual labours
which is more cost efficient.
6) Selective propagation of Diocious Plants.
E.g. Female plants in Papaya and male plants in Asparagus.

19. CALLUS FORMATION IN MEDIUM PLANT GROWN BY MICROPROPAGATION

20. ACHIEVEMENTS
• Raising of whole plant of Lupinus & Tropaelum by shoot tip culture
(1926).
• Virus free Dahlia obtained by meristem culture.
• Commercial propagation of fruits like Apple, Banana etc & acquatic
plants like Ammania & Echinodorus through micropropagation.
• Large scale production of Salvia miltiorrhiza (chinese medicinal
plant).
• Elimination of systemic bacteria from Pelargonium & Diffenbachia.

21. EMBRYO CULTURE
•It is a component of in vitro
fertilization/culture where embryo
excised from immature seeds are
cultured on a synthetic medium. These
cultured embryo can directly develop
into young seedlings.

22. Types of Embryo Culture
1) Mature Embryo Culture : It is the culture of mature embryo derived
from ripe seeds. Eg. Iris & Orchid.
It is done when embryos:
 Do not survive IN-Vitro.
 Become dormant for larger period of time.
 To eliminate the inhibition of seed germination.

24. 2) Immature Embryo culture
 It is the culturing of immature embryos to rescue the embryos
of wild cross.
 It is used to avoid embryo abortion & produce viable plants.
 It requires complex media which includes aminoacids,
hormones, endosperm extract etc.

26. Steps Involved in Embryo
culture
• Seed Coat are soaked in water and cotyledons are splitted open.
• Embryo excision is done aseptically in a laminar air flow cabinet.
Surface sterilization of embryo is generally not done as it is inside seed
coat.
• In case of immature embryo where these are embedded in
endosperm, the incision is made at micropylar end & pressure is
applied to isolate young embryo.
• The excised embryo is transferred to a culture containing nutrient
medium at suitable temperature, photoperiod and humidity.

28. APPLICATIONS
1) Preservation of embryo abortion
- Incompatibility barriers in interspecific and intergeneric
hybridization programmes leading to embryo abortion can be
successfully managed by embryo rescue. E.g. In tribe Triticeae
of grass family Poaceae
2) Overcoming Seed Dormancy
3) Production of Haploid
- Hybridisation of Barley and wheat with Hordeum bulbosum
leading to production of haploid barley and haploid wheat.

30. 4) Overcoming Seed Sterility
E.g. Using embryo culture it is possible to
raise seedling from sterile seeds in Apricots,
Plums & Cherries.
5) Clonal Propagation
- embryo are juvenile in nature with high
regenerative potential, hence they are
favoured for clonal propagation.
6) Propagation of rare plant.
Eg. Colocasia esculentum
7) Rapid seed viability testing.
- Germination of excised embryo is regarded
as more reliable test for determining seed
viability than manual staining method.

31. ACHIEVEMENTS
• Development of resistance in Rice to bacterial blight and blast &
resistance in potato from leaf roll virus.
• Shortening of breeding cycle in plants like rose (6months from 1 year) &
Mallus (5 months from 9 months).
• Barclays (1975) obtained haploid of Triticum aestivum (Triticum
aestivum X Hordeum bulbosum) with the help of embryo culture.

32. OVULE CULTURE
• Ovule culture is an elegant experimental system by which
ovules are aseptically isolated from ovary and grown on
chemically defined nutrient medium or under controlled
condition. It is done when embryo cannot be easily excised.
Ovule culture is a boon for plant breeders in culturing seedlings
from crossed which are normally unsuccessful because of
absorptive embryo.

34. STEPS INVOLVED IN OVULE
CULTURE
•Ovaries are collected and rinsed 3-4
times with sterile distilled water.
•Ovules are prodded with spatula
breaking funicle at junction.
•Ovule is places in sterile solid/liquid
medium for culture. Medium may need
to be supplemented with some fruit or
vegetable juice to accelerate initial
growth.

35. APPLICATIONS
1) For a variety of difficult interspecific /intergeneric crosses
involving members of the families Malvaceae, Fabaceae,
Cruciferae, Solanaceae etc. ovules after fertilisation has been
successfully cultured to obtain mature embryo /seeds.
2) Test tube pollination/Fertilization
• - It is possible to germinate pollen & excised ovule in same culture
to induce in vitro fertilization. E.g. Argemone mexicana , Papaver
somniferum.

36. 3) Production of Haploid Callus
• Uchimiya (1971) cultured unfertilized ovules of Solanum
melongena and obtained callus formation on medium
supplemented with IAA.
4) Induction of Polyembryony in horticultural crops. e.g. Citrus
5) Virus Irradication through Ovule culture im citrus.
6) Orchid seed germinate only in association with fungus but
by culturing fertilized ovules it can grow even in absence of
fungus.

37. ACHIEVEMENTS
• Development of Test tube Pollination & Fertilization through ovule
culture in Argemone mexicana, Papaver somniferum etc.
• Poddubnaya-Arnoldi (1959-60) successfully grew fertilized ovule of
Calanthe veitch, Phalaenopsis schilleriana.
• The hybrid embryo of cross Gossypium barbadense X G. arbarium has
been successfully obtained through ovule culture.

38. OVARY CULTURE
• Culture of unfertilized ovaries to obtain haploid plants from egg
cells is called as Ovary Culture & this process of haploid
production is termed as gynogenesis.
• Growing of ovaries on culture was first done by La Rue in 1942.
• Gynogenesis was first done by San Noem Lu in 1976 in Barley.

39. STEPS INVOLVED IN
OVARY CULTURE
1) Ovaries are excised from flowers in sterile petridish.
2) Ovaries are then places on agar solidified nutrient medium for
culture.
3) Incubate the culture at 25 °C For 1 hour.
Haploid plants mainly emerge from egg cells (in-vitro
parthenogenesis) but in some species (eg. Rice) they arise from
synergids and antipodals (in-vitro apogamy)

40. Applications
1) It helps in study affect of
phytohormones on parthenocarpic
fruits.
2) Problem of slow growth of pollen
tube on stigma can be solved by ovary
culture.
3) It induces Polyembryony.

41. 4) Mature Fruits containing seeds can
be obtained from ovaries excised from
pollinated flowers. E.g. in tomato and
Cucumis.
5) Interspecific hybrids are obtained by
ovary culture.
E.g. B. campestris x B. oleracea.

42. ACHIEVEMENTS
• Development of interspecific hybrid of cross between Brassica
campestris X B. oleracea through Ovary culture.
• San Noem Lu obtained haploid plant of Barley, Wheat & Maize
through Ovary culture.
• In India Prof. P Maheshwari obtained healthy fruits from excised
ovaries in several plants like Iberis amara.

44. Anther Culture
• It is the process of using anthers to culture haploid plantlets.
• This technique was first discovered in 1964 by Guha and
Maheshwari. This technique can be used in over 200 species
including Tomato, Rice, barley etc.
• Haploid production through anther culture is known as
androgenesis.
• Particular Stage of anther at time of culture is important. Usually
anther just before or immediately after pollen mitosis are most
suitable for culture.

45. Suitable stages of anther for culture
are :
1) Pre-mitotic stage
• Anthers at this stage have just completed first meiotic divison and pollens
are immature, uninucleate and starch-free. Anthers of Hordeum vulgare
and Hyocyamus at this stage are suitable for culture.
2) Mitotic Stage
• In some plants like Nicotiana tabacum anthers at first pollen division are
most suitable for culture.

46. 3) Post- Mitotic stage
•Early bi-cellular stage of pollen
development is most suitable for
culture in Atropa belladonna.
•Mature anthers are unsuitable for
culture but in Brassica oleracea,
mature anthers are proper stage for
anther culture.

47. STAGES OF DURAM WHEAT GROWN THROUGH
ANTHER CULTURE

48. STEPS INVOLVED IN ANTHER
CULTURE
•The anthers are carefully excised from flower
buds using forceps and dissecting needle.
•Anther may be cultured either on agar solidified
culture or placed on a filter paper bridge over a
liquid medium. The most popular medium for
Anther culture is N6 Medium.
•Anther are cultured generally maintaining
alternate period of light (12-18 hrs at 28 ° C) and
darkness (6-12 hrs at 22 ° C)

49. • At 3.5cm of height individual plantlets are seperated and
transferred to another medium which support good
development of root system.
• They are then hardened & acclimatized.

51. POLLEN CULTURE
It is a technique in which Haploid
plants are obtained by isolated Pollen
grains.

52. STEPS INVOLVED IN
POLLEN CULTURE
•Anthers are excised from flower buds.
•Anthers are places in a liquid medium. Pollen
grains are removed from anther either
mechanically or by natural dehiscence.
•Float culture technique (discovered by
Sunderland & Roberts) can also be used to
obtain pollen grains from anther. In this method
whole anther is taken from cold treated buds
and are allowed to float on a surface of the
liquid medium. Within few days anther dehisce
and liberate pollen grains at various developmental
stage.

53. • Petridishes containing pollen grains in culture media are sealed
with para film and incubated at 28 °C for 14 days.
• After 14 days culture is kept in illuminated chamber at 25 °C.
• 3-8 weeks are required to obtain haploid plantlets.

54. Factors affecting Anther and
Pollen Culture
• 1) Physiological State of Donor plants like age, response to
environmental conditions etc. Eg. Buds from first flush of flowers
are better for culture.
• 2) Genotype of donor plants. Eg. In Rice Japonica types respond
better than indica types.
• 3) Flowering time - Anther taken from flowers at the beginning of
flowering period is most suitable.

55. 4) Temperature - In Datura embryoid is not formed if temp. is below
20 °C while in Tobacco optimum temperature is 25 °C.
5) The culture medium and conditions during culture Growth. Eg. B5
medium for Brassica and MS, White & N6 media for Solanaceae
crops

56. Applications of Anther & Pollen
Culture
1)Homozygous diploid plants obtained by doubling chromosome of
haploids have great importance in plant breeding.
2) Rapid Production of haploid plants
3) Haploid derived from anther/pollen culture used in cytogenic study.
4) Pollen culture has great significance in mutagenic studies.
5) Selection of Mutants resistant to disease.
Eg. Tobacco Mutants resistant to Black Shank disease.

57. ACHIEVEMENTS
• In Japan an excellent commercial variety (resistant to bacterial wilt)
of Tobacco F-211 has been produced by anther culture.
• In China, 81 varieties of Rice have been developed through Anther
Culture. Eg. Hua yu-1, Xin-Xua etc.
• Tobacco mutants resistant to Black shank disease & wheat lines
resistant to scar has also been developed.

58. Endosperm Culture
• Endosperm tissue is triploid and hence plantlets originating by the
culture of endosperm is also triploid.
• Endosperm tissue is present in vast majority of flowering plant
families except Orchidaceae, Trapaceae & Podostemaceae.
• Immature Endosperm has more potential of growth in culture
especially among cereals.
• Endosperm culture is an easy method for production of large no. of
triploid in one step.

60. STEPS INVOLVED IN ENDOSPERM
CULTURE
1) The immature seeds are dissected under aseptic conditions and
endosperms along with embryos, are excised. Sometimes mature
seeds can also be used.
2) The excised endosperms (with embryos) are cultured on a
suitable medium and embryos are removed after initial growth.
3) The initial callus phase is followed by shoot bud differentiation.
4) The shoots and Roots may subsequently develop and complete
triploid plants can be established.

61. ENDOSPERM CULTURE

62. APPLICATIONS
1) Triploid plants can be raised through
endosperm culture.
2) Endosperm can be used as a nurse
tissue for raising hybrid embryos.
3) Triploid plants (endosperm culture)
are useful for production of seedless
fruits. Eg. Apple, Banana etc

63. 4) To exploit in the biosynthesis of some
natural products. E.g. Cultured
endosperm of Coffee synthesise
caffeine.
5) Triploids raised by endosperm
culture are sometimes even better
than diploids. Eg. Triploids of Populas
have better quality Pulpwood.

64. ACHIEVEMENTS
• Lampe & Mills grew Maize for the first time through Endosperm Culture
in 1933.
• Nikajima obtained mature plants from endosperm of Cucumis sativus.
• Mohan Ram & Satsangi cultured endosperm to obtain mature plants in
Ricinus communis.
• Commercially Important Triploif Plants has also been developed by
Endosperm Culture in species like Banana, Apple, Watermelon etc.

65. THANK YOU