1. TISSUE CULTURE STUDIES IN
CAJANUS CAJAN L.
VIJAY KUMAR CHOUREY
M.Sc.- Genetics (VI Sem.)
DEPARTMENT OF BIOCHEMISTRY & GENETICS
BARKATULLAH UNIVERSITY
BHOPAL (M.P.)

2. Contents
• Introduction
• Material & method
• Result
• Summary
• References

3. Introduction
• Cajanus cajan L. belong to family fabeaceae also known
as red gram, is an important world crop species widely
cultivated.
• Cajanus cajan L. is an excellent source of protein, seeds
are using as vegetable flour additive or other food.
• Cajanus cajan L. flower are self compatible and usually
self pollinated, seed weight 0.1g.
• It forms root nodules is associated with Rhizome sp.
bacteria that function nitrogen fixation.
• 66 chromosome are present in Cajanus cajan L.
(Liogier,at. el., 1988 )

4. • It is usually single stemmed, freely branching, and
become woody after a few months.
• India is the world’s largest pigeonpea producer and
grows over 77% of the total world production.
• Typical nutritional values for seeds are: moisture, 10.1
percent, protein 19.2 percent, fat, 1.5 percent,
carbohydrates, 57.3 percent, fiber 8.1 percent, and ash,
3.8 percent (Smartt 1976).
• Hence, present study was carried out with the following
objectives:
- To study of different explant give rise to callus
induction.
- To study of redifferentiation of in Cajanus cajan L.

5. Material & Methods
MATERIAL
• Seeds
Healthy seeds of Cajanus cajan L. variety Balwan (NRS)
used in the present study were obtained from Mukesh
seeds, Bhopal.
• Glassware
Glassware used in the present study was obtained form
“Borosil” India. Glass bottles, test tubes, petriplates,
beakers, conical flask etc. were used in the study.
• Chemical
All the chemicals used in the present study were analytical
grade and were obtained from CDH, Himedia and Qualigens
India. Plant growth regulators, gelling agent, sucrose,
vitamins, colchicine etc, were obtained from “CDH fine
chemicals” and “Himedia” Mumbai.

6. Method
• The tissue culture laboratory was washed with effective
disinfectant (Teepol) followed by wiping with 2% sodium
hypochlorite solution or 95% ethyl alcohol, extran, Lysol,
zephiran or roccal.
• 1L. MS, White and B5 media are used
– 30 g (3% w/v) sucrose,
– Required quantities of plant growth regulators &
– Micronutrient and Macronutrient were added,
– 8 g agar,
– Make up final volume (i.e. 1000ml),
– pH was adjusted between 5.2-5.8,
–
• Media was boiled for dissolving agar and dispensed in culture
bottle & culture tube.
• Finally, the culture vessels were plugged, labeled and
autoclaved at 1210
C and 1.06 kg/cm2 for 20 min.

7. • Surface Sterilization of Seeds:
Different varieties seeds were surface sterilized by
washing with tap water for several times to remove soil or
dust deposits followed by a treatment with 70% (v/v)
ethanol for 1 min and rinsing with sterilized distilled
water for thrice.
0.1% mercuric chloride solution use for five minutes and
finally rinsed with sterilized distilled water for 4-5 times in
order to remove all the traces of surfactants. Sterilized
seeds thus obtained were used for in-vitro seed
germination.
• In- vitro Seed Germination
Sterile seeds of Cajanus cajan L. accession Balwan (NRS)
were inoculated in different basal media, namely MS
(Murashige and Skoog 1962), B5 (Gamborgs et. al., 1968)
and WH (Whites, 1963).
• Explants Selection
The present study, seeds, apical and axilliary buds,
cotyledonary leaves and hypocotyl parts were used as
explants.

8. Result
• Seed germination
The seed germination potential of Balwan in MS, White and B5 plain medium
in one week was found highest with MS medium (93.30%) followed by White
(83.30%) and B5 (70.01%).
• Table-1 Seed germination potential of Cajanus cajan L. in MS, WH and B5
media (plain)
Medium No. of seeds Seed germination Average length of seedling (cm)
after 7 days (%) 7 days* 21 days*
MS 25 93.30% 4.2 ± 0.15 9.5 ± 0.21
WH 25 83.30% 3.2 ± 0.12 7.2 ± 0.18
B5 25 70.01% 3.1 ± 0.12 6.7 ± 0.17
Note: * Based on replicates of five seeds each.

10. In vitro seed
germination in B5,
WH and MS media
after 7 days.
In vitro seed germination on
MS medium after 21 days.

11. Table-2 Callus induction and redifferentition response with different
Axillary Shoot explants of Cajanus cajan L. in MS medium
supplemented with variable concentrations of growth adjuvants.
Explant
MS + Growth adjutants
(mg/l)
14 days 21 days
NAA BAP
Callus
induction
(%)
Redifferentiatio
n Response (%)
Callus
formation in
Explants*
Axillary
Shoot
0.25 0.25 0.00 0.00 0.00
0.50 0.25 40.00 16.00 +
0.75 0.25 60.00 44.00 ++
1.0 0.25 66.00 50.00 ++
1.5 0.25 46.00 37.00 +
2.0 0.25 20.00 31.00 +
Note:* Relative value of Callus formation on Explants: -, Nil; +, Slight; ++, Moderate; +++, High.

12. Explant
MS + Growth
adjuvants (mg/l)
14 days 21 days
NAA BAP
Callus
inductio
n (%)
Redifferentiati
on Response
(%)
Callus
formation
in
Explants*
Hypocotyl,
0.25 0.25 40.00 40.00 +
0.50 0.25 53.00 43.00 ++
0.75 0.25 58.00 53.00 +++
1.0 0.25 86.00 68.00 ++
1.5 0.25 60.00 57.00 ++
2.0 0.25 43.00 34.00 +
Table-3 Callus induction and redifferentition response with Hypocotyl
explants of Cajanus cajan L. in MS medium supplemented with
variable concentrations of growth adjuvants.
Note:* Relative value of Callus formation on Explants: -, Nil; +, Slight; ++, Moderate; +++, High.

13. Table-4 Callus induction and dedifferentiation response with Stem
explant of Cajanus cajan L. in MS medium supplemented with variable
concentrations of growth adjuvants.
Explant
MS + Growth
adjuvants (mg/l)
14 days 21 days
NAA BAP
Callus
inductio
n (%)
Redifferentiati
on Response
(%)
Callus
formation
in
Explants*
Stem
0.25 0.25 20.00 23.00 +
0.50 0.25 33.00 26.00 +
0.75 0.25 40.00 32.00 ++
1.0 0.25 60.00 44.00 +
1.5 0.25 41.00 33.00 +
2.0 0.25 26.00 25.00 +
Note:* Relative value of Callus formation on Explants: -, Nil; +, Slight; ++, Moderate; +++, High.

14. Explant
MS + Growth adjuvants
(mg/l)
14 days 21 days
NAA BAP
Callus
induction
(%)
Redifferentiatio
n Response (%)
Callus
formation in
Explants*
Cotyledonary
leaves.
0.25 0.25 0.00 0.00 -
0.50 0.25 48.00 25.00 +
0.75 0.25 46.00 28.00 +
1.0 0.25 52.00 36.00 ++
1.5 0.25 38.00 32.00 +
2.0 0.25 33.00 26.00 +
Note:* Relative value of Callus formation on Explants: -, Nil; +, Slight; ++, Moderate; +++, High.
Table-5 Callus induction and dedifferentiation response with
different Cotyledonary leaves explants of Cajanus cajan L. in MS
medium supplemented with variable concentrations of growth
adjuvants.

15. • Callus induction and redifferentition response with different
explants of Cajanus cajan L. in MS medium supplemented with
variable concentrations of growth adjuvants.

16. Cotyledonary
leaf
Hypocotyl
Axillary shoot
Callus induction

17. Table 6:Optimum concentration of cytokinin alone in
subculture for multiplication and elongation of shoots raised
intervening by callus in Cajanus cajan L.
Shoots (in 21 days)
Explant MS+ BAP
(mg/l)
Shoot
multiplicatio
n response
(%)
Average no.
of* shoots
(Mean ±
S.E.)
Average
length* of the
multiplied
(Mean ± S.E.)
Callus raised shoot
from hypocotyl
0.25 40 3.91 ± 0.39 2.03 ± 0.21
0.50 52 4.33 ± 0.50 2.66 ± 0.17
0.75 55 6.43 ± 0.34 3.08 ± 0.18
1.0 75 13.33 ± 0.41 5.33 ± 0.21
1.25 64 8.58 ± 0.53 4.33 ± 0.23
1.50 44 4.8 ± 0.67 3.00 ± 0.24
Note: * Based on six replicates of five explants each.

18. • Shoot multiplication from callus obtained from hypocotyls explant.
Shoot multiplication

19. Table 7: Root induction with auxin in elongated adventitious
raised from hypocotyl in Cajanus cajan L.
Explant
½ MS media +
Growth regulator
(mg/l)
Roots (after 21 days)
Root
Induction
(%)
Mean no. of*
root ± S.E.
Mean root*
length ±
S.E.
Elongated
shoot
through
callus
½ MS plain 38 2.30 ± 0.20 0.65 ± 0.02
IBA (0.25) 62 2.50 ± 0.24 0.80 ± 0.04
IBA (0.50) 66 3.66 ± 0.30 1.22 ± 0.15
IBA (0.75) 70 5.50 ± 0.24 1.30 ± 0.08
IBA (1.00) 72 5.70 ± 0.32 1.50 ± 0.06
IBA (1.25) 85 6.50 ± 0.34 2.85 ± 0.06
IBA (1.50) 34 3.75 ± 0.31 1.25 ± 0.05
Note: * Based on five replicates of three explants each.

20. Fig.: Root induction in callus raised shoots obtained from hypocotyls explant.
Root induction

21. Summary
• The present project report entitled “Tissue Culture Studies in
Cajanus cajan L.” presents findings and inferences of tissue
culture experiments carried out through different organ
explants in order to demonstrate a reproducible protocol for in
vitro regeneration in Cajanus cajan L.
• Seeds, apical and axillary node, cotyledonary leaves and
hypocotyl parts were used as explants.
• In present investigation all cultures were exposed to constant
intensity of light (2500 Lux) with photoperiod of 16 h light/ 8 h
dark at 25 ±2 0 c provide short-day environment (8 h light/ 16 h
dark).
• Present study principally focuses on regeneration through
organogenesis and demonstrates organogenesis competence
of hypcotyl portions in Cajanus cajan L., chosen for the study.

22. • Seed germination potential in different media involving MS, WH
and B5 was noted to be highly variable in different set of
experiments. MS basal medium (full strength), and supplemented
with different concentrations of BAP. Seed germination potential of
different cultivars of Cajanus cajan L. in MS basal media
supplemented with similar concentrations of BAP has not shown
considerable variation.
• Highest redifferentiation response noted (68%) with 1.0 NAA
mg/l and 0.25 mg/l in hypocotyls explant.
• The highest shoot multiplication response was noted with 1.0
mg/l BAP supplemented to full strength MS medium.
• The highest root induction at 85% was noted with 1.25mg/l IBA.

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26. Thank you