TISSUE CULTURE STUDIES INCAJANUS CAJAN L.

VIJAY KUMAR CHOUREYM.Sc.- Genetics (VI Sem.)

DEPARTMENT OF BIOCHEMISTRY & GENETICSBARKATULLAH UNIVERSITY

BHOPAL (M.P.)

Contents

• Introduction

• Material & method

• Result

• Summary

• References

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 )

• 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.

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.

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 1210C and 1.06 kg/cm2 for 20 min.

• 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.

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.

In vitro seed germination in B5, WH and MS media after 7 days.

In vitro seed germination on MS medium after 21 days.

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 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.

Explant

MS + Growth adjuvants (mg/l)

14 days 21 days

NAA BAPCallus induction (%)

Redifferentiation 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.

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 BAPCallus induction (%)

Redifferentiation 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.

Explant

MS + Growth adjuvants (mg/l)

14 days 21 days

NAA BAPCallus induction (%)

Redifferentiation 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.

• Callus induction and redifferentition response with different

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

Cotyledonary leaf

Hypocotyl

Axillary shoot Callus induction

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 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.

• Shoot multiplication from callus obtained from hypocotyls explant.

Shoot multiplication

Table 7: 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

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.

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

Root induction

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.

• 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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