about tissue culture

2. UNIVERSITY OF HORTICULTURAL SCIENCES
, BAGALKOT.
PRESENTATION
ON
Tissue culture and Biotechnological approaches in
Plantation crops
BY:
Mr. CH. Lokesh
UHS14PGM433
KITTUR RANI CHANNAMMA COLLEGE OF
HORTICULTURE, ARABHAVI

3. Tissue Culture
Tissue culture is a technique of growing plant cells by
culturing ex-plant aseptically on a suitable nutrient
medium.
Tissue Culture technique relies on:
 Ex-plant
 Aseptic environment
 Nutrient media.
Modes of Culture:
(i)Callus culture
(ii)Cell suspension culture
(iii)Single cell culture.

4. Biotechnology:
Biotechnology is name given to the methods and
techniques that involve the use of living organisms
like bacteria, yeast, plant cells etc or their parts or
products as tools (for example, genes and
enzymes)
Plant tissue culture comprises a set of in vitro
techniques, methods and strategies that are part of
the group of technologies called plant
biotechnology.

5. Applications of biotechnology:
Micro propagation of biomass energy production.
Production of disease free, disease resistant,
insect resistant, herbicide resistant plants.
Induction and selection of mutants.
Production of somatic hybrids.
Production of somatic plants.
Industrial biotechnology.

6. Coconut
 S.N: Cocos nucifera
 Family: Palmae
 Chromosome no:2n=32.
 Is a versatile palm with varied uses.
 It is also called as Kalpa vriksha,
Tree of heaven.

7. Micro propagation:
 Current production of quality planting materials meets only
20% of estimated annual requirement of planting material
to replace senile and disease free plantation.
 In vitro vegetative multiplication through somatic
embryogenesis of high performance individuals thus offers
the only hope for production of homogeneous planting
materials and for substantial improvement in plantation
productivity.

8.  Unfortunately coconut is highly recalcitrant sp with respect
to tissue culture.
 success in this area has been limited and only a few clonal
plants have been ever established in the field.
Various problems encountered during in vitro propagation of
coconut are….
1.Intensive tissue browning (due to oxidation of polyphenols),
2. Slow in vitro response,
3.low rate of somatic embryogenesis,
4.variation in tissue response due to heterogeneity of explants
taken from different individuals.

9. A variety of protocols have been developed using a range of
explants:
• Immature inflorescence,
• Immature and mature zygotic embryos,
• Young tender leaflets, leaf bases from unopened spindle
and plumular tissue.

10. Embryo culture
• Embryo culture has become an important tool for safe
Germplasm movement.
• A simple protocol of culturing coconut embryos of 8 to
11month-old nuts was standardized and successfully
utilized in Germplasm expeditions.

11. Protocol broadly consisted of four components:
I. Field collection of embryos.
II. In vitro active storage.
III. In vitro retrieval and
IV. Ex vitro establishment of plantlets.
• Embryo, along with a portion of endosperm, is scooped
out, surface-sterilized, washed and directly inoculated.
• In vitro active conservation (short-term storage) of
coconut zygotic embryos is ideal for enabling embryo
storage (for up to 2 months in sterile water).

12. • For ex vitro studies, protocol uses a potting mixture
consisting of sterile soil, sand and coir dust in equal
proportions.
• Before transplanting to pots, leaves are trimmed and
seedlings are treated with Bavistin (1 g/L) and thereafter
with IBA solution (1000 ppm) as a precautionary measure.
• To adjust humidity conditions, plantlets are covered with
polythene bags that are gradually perforated.

14. • Zygotic embryo culture was utilized for Germplasm
expeditions of coconut.
• Percent retrieval of embryos varied among location and
among accessions.
• A major cause of differential germination was
contamination of cultures.
• Treatment of cultures with tetracycline (2 ppm) is effective
in treatment of cultures with mild bacterial contamination.
• Germination (%) varied between 54 to 82.2.

15. • Observations on in vitro retrieval of embryos and their
ex vitro establishment suggest that, about 300 to 400
embryos per accession needed to be collected for field
establishment of 100 plants in a gene bank.

16. In vitro conservation
• Coconut genetic resources are traditionally conserved ex
situ in gene banks.
• Use of in vitro culture techniques including slow growth
and cryopreservation, represents an important
additional option for safe medium and long term
conservation of coconut Germplasm.
• A medium containing 2g/l of activated charcoal could
store embryos for 6 months and gave 77% germination.

17.  Immature embryos from nuts of 7-8 months after
pollination could be successfully cryo preserved and
retrieved.
 Embryos were desiccated for 4 h in air current of laminar
flow cabinet, pre-treated for 11-20 hrs on a medium
containing 600 g /liter sucrose and 15% glycerol and then
rapidly immersed in liquid nitrogen.
 Freezing and thawing with recovery rates between 33
and 93% of frozen embryos, depending on the variety was
reported.
 Subsequently optimal conditions for the medium-term
conservation of zygotic embryos were developed.

18. • Immature embryos collected from 10 and 11 months
old nuts of two exotic Germplasm viz., Cochin China
Tall (CCNT) and Malayan Yellow Dwarf (MYD) cultivars
of coconut were utilized for study.
• Extracted embryos were stored (30 days) individually
in 5 ml screw capped bottles containing 2 ml of storage
medium.

19. • Germination at 60 days after inoculation was significant
with type and age factor.
• Regarding type, germination was more Dwarf type
(93.7%) whereas for CCNT it was 85.6%.
(Brian, 2006.)

20. What makes clonal propagation of coconut
difficult?
S.C. Fernando*, V.R.M. Vidhanaarachchi, L.K. Weerakoon
and E.S. Santha
Tissue Culture Division, Coconut Research Institute,
Lunuwila, Sri Lanka
Proceedings Asia Pacific Conference on Plant Tissue and Agribiotechnology
(APaCPA) 17-21 June 2007

21. Some of the major constraints are…
1
2
Heterogeneous response of various
explants due to the influence of ex-plant
maturity and genotype.
Undefined culture conditions with regard to the
hormonal composition of the culture medium
due to the presence of activated charcoal.

22. 3
4
5
Production of highly heterogeneous and
compact colloids due to partial de-
differentiation of cells of explants.
Poor plant regeneration efficiency due to
marked influence of genotype and variable
culture conditions.
Slow growth of tissue-cultured plants under in
vitro and early ex vitro conditions.

23. How to overcome these limitations?
1 Selection of the most responsive ex plant
2
3
4
5 Application of stress conditions to improve
somatic embryogenesis and plant regeneration
Development of a charcoal-free medium
Establishment of cell suspensions
Repeated multiplication of embryogenic
cell masses

24.  Activated charcoal is an essential component of coconut tissue
culture medium.
 It has strong adsorptive properties and its beneficial effects are
attributed to the adsorption of phenols and other growth inhibitory
substances.
 However, a major disadvantage of using activated charcoal is that
it also can adsorb plant regulators (hormones, vitamins) and some
minerals (Cu and Zn) (Pan and Staden, 1998).
Effect of activated charcoal
As a solution, a charcoal-free medium was developed…
Addition of PVP (20 gr/l) and ascorbic acid to culture
medium in place of charcoal gave rise to 60 % callus.
 However, the embryogenic potential of those calli was low (Diyasena,
1998).
 Use of a single type of charcoal under controlled conditions could be a
way to overcome the problem.

25. Rubber
• S.N: Hevea brasiliensis.,
• Family: Euphorbiaceae
• Origin :Amazon river basin of South America
• The principal source of natural rubber.
• It is intensively cultivated in South East Asia.

26. Micro propagation
• Propagation of rubber is possible through tissue culture also.
• Studies on tissue culture of rubber plants were started in
1966.
• Different parts of the plant such as embryo, anther, shoot
tip and integument can be used for tissue culture.

27. • Rubber Research Institute of India has developed a
technique for the production of tissue culture plants from
shoot tips as well as somatic embryogenesis of different
tissues.
• Rubber plants were developed by the somatic
embryogenesis of anther tissue, Integumental tissue,
immature Inflorescence etc.
• Attempts are being made for the tissue culture of other
plant parts like leaf, floral buds, ovules and micro spores.

28. Key steps in tissue culture of Hevea include :
 collection of the explant.
 sterilization, inoculation of the explant in a nutrient media
supplemented with growth hormones and sucrose.
 The cultures were kept under optimum light and
temperature conditions for the required period.
 Plants were formed in about eight months.
 They were then transferred to small poly bags and kept in a
green house for hardening.
 Even for the same clone the culture conditions vary with the
physiological stage of the explant, seasons, part of the plant
used etc.

29. Somatic embryogenesis:
• Somatic embryogenesis is one of the powerful tissue
culture techniques for mass propagation of elite Hevea
clones.
• High frequency somatic embryo induction and plant
regeneration were achieved from immature anthers and
inflorescence.
• Optimum callus induction was obtained in modified MS
medium supplemented with 2.0 mg/L 2,4-D and 0.5 mg/L
Kinetin.

30. • Immature anthers pre-cultured in liquid medium for 10
days followed by 25 days culture in solid medium
promoted callus induction.
• Embryo induction efficiency was promoted by
supplementing 200 mg glutamine and 400 mg casein
hydrolysate in embryo induction medium.
• Incorporation of GA3 up to 2.0 mg increased embryo
induction frequency.

31. • Good callus induction and high frequency somatic
embryogenesis were obtained using leaf and ovule as
explants.
• Maturation, germination and bipolar differentiation of
embryos were achieved in MS medium devoid of growth
regulators.

32. Callus initiation
from immature
anther
explants.
Embryogenic
callus formation
Different stages of
somatic embryos

33. Somatic embryo derived
plantlets
Embryo germination
and plantlet
development
Somatic embryo
derived plants

34. Anther culture
• Rubber being a perennial tree crop with a long life span
and the available varieties of the rubber trees are all
highly heterozygous.
• Homozygous diploid pollen plantlets could be directly
utilized in crop improvement programmes.
• The possession of haploid and double haploid plants, on
which both dominant and recessive characters would be
expressed, considerably shortens the process of
selection of desirable characters.

35. • The heterosis of good combinations of pure lines is much
higher than that of crosses between two different
varieties.
• Since 4-5 years are required for Hevea from sowing to
flowering and the fruit set success on selfing is very low,
it is highly impossible to obtain pure lines by
conventional breeding.
• By the establishment of anther culture and production of
haploid plants one can develop pure lines of different
genotypes on a short time and thus pave the way to
produce uniform sexual lines combining desirable
characters (Chen, 1984; Asokan et al., 1992)

36. • Das et al. (1994) studied the effect of low temperature a
on somatic embryogenesis from anther-derived calli.
• Anthers of various clones were cultured on different
media.
• High sucrose (6% ) in the medium and cold treatment of
anthers at 8-10° C for 24 hours had positive effects on
callus induction.
• Supplementation of the medium with benzyl
adenine(BA) along with combination of IAA or NAA
produced the highest number of somatic embryos.

37. Protocol for anther culture
The protocol reported by Chen (1984) is as follows for the
induction of pollen plantlets.
It is carried out in three steps :
I. Anther is inoculated on primary medium to induce callus
formation in which the microspores grow and develop
into multi-cellular masses, haploid embryos or calli.

38. II. The pollen-derived callus is transferred to differentiation
medium in which they develop into minute embryos which
subsequently develop into the embryos visible to the naked
eye.
• At this point, about 70% of the cells have the haploid
chromosome number.

39. III. The well developed microspore embryos are transferred
to plantlet forming medium on which they further
develop into intact plantlets.
• For callus induction a medium containing macronutrients
and iron salts of MS medium with reduced nitrate and
micronutrients, vitamins and organic supplements as
suggested by Bourgin and Nitsch (1967) with 5% coconut
water containing 9.3 M Kinetin and 9.6 M 2,4-D.

40. • During the process of embryogenesis, different
morphological types, such as round, trumpet, rod and
cotyledon types were observed of which the trumpet and
cotyledon types are found to be more normal producing
majority of plantlets.
• Once the microscopic embryos are formed in the
differentiation medium, it will take about 2-3 months to
develop terminal buds.
• The embryos should be transferred to plantlet formation
medium only after development of terminal buds.
• Otherwise, the embryos form roots but not shoot

41. Invitro Conservation
• Conservation of genetic resources of Hevea is an urgent
need of the time.
• International Plant Genetic Resources Institute (IPGRI),
formerly International Bureau of Plant Genetic Resources,
(IBPGR) has included rubber as one of the highest priority
crops for conservation of the entire gene pool (IBPGR,
1984).
• Both in situ conservation of genotypes in their original
habitats and ex situ conservation in special nurseries or
fields are feasible in Hevea, of which the latter is widely
adopted due to practical considerations.

42.  The storage of Hevea seeds to prolong seed viability is
important because seeds are used for the production of
seedling rootstocks and as planting material.
 In vitro conservation of Hevea accessions, through
cryopreservation has been suggested as a viable option
for the long term storage of germplasm.
 Though cryopreservation protocols have been developed
for various field crops, attempts in this direction are also
in Hevea.

43. • Recently two efficient cryopreservation protocols, were
developed for embryogenic calli of a commercial clone of
Hevea, (PB 260).
 Using a classical freezing process
 Simplified freezing process.

44. • After pre culture with 1% sucrose and 10% di methyl
sulphoxide, embryogenic calli were frozen in a
programmable freezer at 0.2°C down to -40°C.
• So that High survival and rapid re growth, as well as
production of somatic embryos, were observed.

45. Oil palm
S.N: Elaeis gueineensis
Family : Palmae
Origin: West Africa
Chromosome no:2n=32
Cheapest and major oil yielding crop.

46. Biotechnology
Objectives:
• Producing good tenera palms for commercial plantings.
• To multiply parents (both dura and pisifera) for seed
production.
• To exploitation of genetic potential of progenies from
interspesific E. oleifera X E. gueninsis crosses
• To salvage diseased palms .

47. Tissue culture process :
• Leaves, Inflorescenses , and roots are used as explants for
oil palm tissue culture.
• Young leaf spears are preferred in most laboratories.
• Leaf explants can be surface sterilized easily, and give
higher clonability rates.

48. The process of oil palm tissue culture can be divided into
several different stages.
1.Callus is initiated from the explant.
2. Embryogenesis
3 shoot and root regeneration,
4.hardening of plantlets
5.Field evaluation

49. • The regeneration process through oil palm takes 2 or 4
years depending on genotype.
• Growth conditions for different stages are typically at 28°C
± 2°C with equal light and dark.
• Explants are placed in media containing either 2, 4-D or
NAA for 12 to 14 weeks callus formation.
• The calli are maintained in media containing lower
concentrations of 2, 4-D or NAA for up to 12 months for
multiplication and embryogenesis.

50. • The rates of callusing and embryogenesis in oil palm were
demonstrated to be genotype dependent .
• A few oil palm clones produce embryoids after one
month, whereas others may take as long as 24 months.

51. • Small clumps of poly embryoids are transferred onto a
basal nutrient medium and kept for at least three months
for shoot induction.
• Shoots obtained are separated from poly embryoids and
placed onto solid shoot development media containing
low concentrations of NAA in culture tubes (takes two to
three shoots) or flasks (about 15 shoots).
• When the shoot height reaches 5 Cm, they are transferred
into liquid root-initiation media.

52. Zamzuri .,(1998), introduced double-layer rooting technique
for oil palm.
• In this technique, solid shoot development medium is
over layed with liquid root initiation media.
• The plantlets are transplanted into small polybags
containing 1: 1 ratio of soil and sand and kept for three to
four months under shade with relative humidity of more
than 70% for acclimatization before transferring to field
nursery.
• In the nursery, plantlets are look like seed derived
plants.

53. • In india plantlet develepment was reported from leaf
explants of 18 months old dura and 6 months old tenera
oil palm seedlings.

54. Cryopreservation:
 The need to conserve oil palm in in vitro arises because of the
large area which would be needed for ex vitro field
conservation.
 Very little work has been done on this aspect inspite of its
importance.
Engelmann et al. (1988) were the first to standardize the
technique of cryopreservation.
 They were able to store embryoids for 15 months in liquid
nitrogen and then could regenerate plantlets from the frozen
materials.

55. CASHEW NUT
• S.N: Anacardium occidentalae
• Family: Anacardiaceae
• Origin: Brazil
• Chromosome No:24 to 42.
• Is an export oriented crop grown for its nuts.
• It is also called as dollar earning, plough crop.

56. Biotechnology
• Areas where biotechnology would be of importance in
cashew are somatic embryogenesis and plantlet
regeneration.
• It is useful for genetic transformation to introduce genes
for resistance to tea mosquito bug, stem and root
borers.
• Standardization of micro-grafting technique, developing
haploids and isogenic lines and molecular
characterization of existing genetic diversity.

57. micro-propagation
• Micro propagation using seedling explants was
standardized.
• Cotyledon explants produced 10-12 shoots/ culture and
shoots were rooted by exposing to IBA (20-60 ppm) for 2
hours.
• Regeneration from mature tree explants has been difficult
due to high rate of contamination, browning, slow growth
and poor rooting of micro shoots.
• plantlets were regenerated from shoot explants taken
from 2-3 year old grafted plants, which were regularly
sprayed with Bavistin (0.1%) at weekly intervals.

58. • Among different surface sterilization treatments used, a
combination of antioxidants, antibiotics and fungicides was
better with around 40 % contamination free cultures being
obtained.
• MS medium containing full strength macro elements
supplemented with Kinetin (5 mg /L), NAA (1 mg /L) and
Brassinolide (0.1 mg/L) was the best for shoot proliferation
and shoot elongation.

59. • Micro shoots were rooted in vitro at a frequency of 70-80
%, when cultured for 4-8 days in quarter strength MS
liquid medium supplemented with IBA (1 mg/L) .
• About 50-60 % of rooted micro shoots survived and
produced healthy and vigorously growing plants, which
were transferred successfully to the field.

60. Micro grafting :
• A successful micro grafting technique to rejuvenate mature
cashew tree explant material was developed using in vitro
germinated seedling as root stock and shoots from stage II
cultures as scions.
• Grafting success depended on method of grafting, size of
scion and age of rootstock.
• Among methods tried, side grafting preferred, due to ease
of operation and faster union.

61. • High levels of graft union were obtained when 2-3 cm
long shoots from stage II cultures were grafted on to 8-9
days old in vitro raised root stocks in full strength MS
medium supplemented with Kinetin (2.5 mg/L), NAA (0.5
mg /L) and Brassinolide (1 mg/L).
• Micro grafts after a process of hardening could be
successfully transplanted into soil.

62. Embryo culture :
• Embryos from immature nuts of cashew were cultured
in vitro to regenerate improved hybrid plantlets.

63. • Explants were surface-sterilized.
• Aseptically dissected.
• Cultured into pure basal MS agar medium and MS
medium supplemented with 1 micro M each of NAA,
benzyl adenine (BA) and Gibberellic acid (GA3).
• Subsequently observed for germination and survival
rates until successful ones were transferred to field.

64. • Explants 6 weeks old or older show better germination
rate and the highest survival percentage.
Factors affecting germination Of embryo:
 Medium composition,
 Age of embryo and
 Genotype .
• Germinated explants simultaneously produced shoots and
roots, and were ready for transfer to field and
acclimatization between 90 and 112 days after inoculation.

65. Somatic embryogenesis and plantlet
regeneration from leaf and
inflorescence explants of Arecanut
(Areca catechu L.)
Anitha Karun*, E. A. Siril, E. Radha and
V. A. Parthasarathy
Biotechnology Section, Central Plantation Crops Research Institute,
Kudlu P.O., Kasargod 671 124, India.

66. • Application of tissue culture technology in clonal multiplication
of oil palm, date palm, and coconut has been reported, but not
in Arecanut except for a report on adventitious shoot
development from mature embryos.
• In vitro retrieval of Arecanut embryos was reported to be
Successful.

67. In vitro propagation of Arecanut through inflorescence
explant
(var. Sumangala)
A: Callus development from inflorescence segment
cultured in MS medium containing Picloram.
(Picloram was found to be the most suitable
callogenic agent)
B:Embryogenic callus developed in Picloram-added
medium.

68. C: Fully developed somatic embryos.
D: Germination of somatic embryos and meristemoid formation.
E: Plantlet development through meristemoids in BA-added
medium.

69. F: Somatic embryo-derived plantlet growing in half-strength
MS liquid medium containing BA (5 mM).
G: Hardening of in vitro raised areca plantlet.
H: Somatic embryo-derived plant growing in
shade net-house.
Anitha Karun et., 2004

70. Limitations in tissue culture teqnique….
1. Soma clonal variations,
2. Difficulties in rooting, browning of media due to phenolic
compounds,
3. Fungi, bacteria prasent with in the plant tissues,
4. Requirement of highly equipped labs, skilled persons..

71. Venkatachalam, P., et al. 2007 , Current Perspectives on Application of
Biotechnology to Assist the Genetic Improvement of Rubber Tree
(Hevea brasiliensis Muell. Arg.)
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Saenz, L., 2006, Pollen Fertility and Female Flower Anatomy of
micropropagated coconut palms. Vol:29.004, Mexico; 373-8.
Anitha Karun*, E. A., Siril , E., Radha and V. A. Parthasarathy, 2004,
Somatic embryogenesis and plantlet regeneration from leaf and
inflorescence explants of arecanut (Areca catechu L.). CURRENT
SCIENCE, VOL. 86, NO. 12, 25.
Bavappa, K. V. A., Nair, M. K. and Premkumar, T., 1982,
The Arecapalm (Areca catechu Linn.). Central Plantation Crops
Research Institute, Kasargod,

72. Rajesh, M. K., Radha, E., Anitha Karun and Parthasarathy, V. A., 2003,
Plant regeneration from embryo-derived callus of oil palm – the effect of
exogenous polyamines. Plant Cell Tiss. Org. Cult.,75, 41–47.
Rethinam, P. and Sivaraman, K., Arecanut (Areca catechu L.):
present status and future strategies. 2000 ,Indian J. Arecanut, Spices
Med. Plants,, 3, 35–50.
Karun, A. and Sajini, K. K., 1996, Plant regeneration from leaf
explant of oil palm. Curr. Sci.,, 71, 922–926.