1. VIJAY KUMAR CHOUREY
DEPARTMENT OF BIOCHEMISTRY & GENETICS
BARKATULLAH UNIVERSITY
BHOPAL (M.P.)
MULITIPLESHOOT INDUCTION AND
PLANT REGENERATION IN
PIGEONPEA (CAJANUS CAJAN L.)

2. Contents
Introduction
Materials & methods
Results
Conclusion
References

3. INTRODUCTION

4. Introduction
Cajanus cajan L. belong to family fabaceae 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.
Its flower are self compatible and usually self pollinated.
Its forms root nodules associated with Rhizobium sp.
bacteria that function nitrogen fixation.
66 chromosome are present in Cajanus cajan L.

5. 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.

6. Present study was carried out with the following
objectives:
1. To study of different explant for to
callus induction.
2. To study redifferentiation in Cajanus
cajan L.

7. METERIALS
AND
METHODS

8. Material & Methods
MATERIAL
 Healthy seeds of Cajanus cajan L. variety Balwan (NRS) used
in the present study were obtained from Mukesh seeds,
Bhopal.
 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.

9. 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.
 MS medium used for present study:
 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 5.4- 5.6.
 Finally, the culture vessels were plugged, labeled and
autoclaved at 1210
C and 1.06 kg/cm2 for 20 min.

10. Explants Selection
 The present study, apical, axilliary buds, cotyledonary
leaves and hypocotyl parts were used as explants.

11. RESULTS

12. Table-1 Callus induction and redifferentiation 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.

13. 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 +
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 +

14. 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.

15. Fig. 1: Callus induction and redifferentiation response with different explants of
Cajanus cajan L. in MS medium supplemented with variable concentrations of
growth adjuvants.

16. Fig. 1a: Callus induction from Hypocotyl parts

17. Table 2: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
multiplication
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. Fig 2: Shoot multiplication from callus obtained from hypocotyls
explant.
Shoot multiplication

19. Table 3: 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
derived
shoots
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.3: Root induction in callus raised shoots obtained from hypocotyls explant.
Root induction

21. CONCLUSION

22. Conclusion
 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.
 Present study principally focuses on regeneration through
organogenesis and demonstrates organogenesis competence of
hypcotyl portions in Cajanus cajan L., chosen for the study.

23. References
Blümmel, M and Saxena, KB (2005): Genetic variation for forage
quality parameters in pigeonpea. Fourth International Food Legumes
Research Conference, New Delhi, October 18–22, 2005
Butler, JE (1910): The wilt disease of pigeonpea and the parasitism of
Neocosmospora vasinfecta Smith., India. Dept. Agr. Memoir. Bot. Ser
11(9):1–64.
George, L; Eapen, SL (1994): Organogenesis and embryogenesis from
diverse explants in pigeonpea (Cajanus cajan L.). Plant Cell Rep., 13:
417-420.

24. Thank