VIJAY KUMAR CHOUREYDEPARTMENT OF BIOCHEMISTRY & GENETICS

BARKATULLAH UNIVERSITYBHOPAL (M.P.)

MULITIPLESHOOT INDUCTION AND PLANT REGENERATION IN PIGEONPEA (CAJANUS CAJAN L.)

Contents

IntroductionMaterials & methodsResultsConclusionReferences

INTRODUCTION

IntroductionCajanus 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.

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.

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.

METERIALS AND

METHODS

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.

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 1210C and 1.06 kg/cm2 for 20 min.

Explants Selection

The present study, apical, axilliary buds,

cotyledonary leaves and hypocotyl parts were

used as explants.

RESULTS

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 BAPCallus induction (%)

Redifferentiation 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.

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 +

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.

Fig. 1: Callus induction and redifferentiation response with different explants of

Cajanus cajan L. in MS medium supplemented with variable concentrations of growth adjuvants.

Fig. 1a: Callus induction from Hypocotyl parts

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.

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

Shoot multiplication

Table 3: Root induction with auxin in elongated adventitious raised from hypocotyl in Cajanus cajan L.

Explant½ 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.

Fig.3: Root induction in callus raised shoots obtained from hypocotyls explant.

Root induction

CONCLUSION

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.

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.

Thank you