Indian Journal of Biotechnology Vol 4, October 2005, pp 556-560

Callus induction and organogenesis from various explants in Vigna radiata (L.) Wilczek Srinath Rao*, Prabhavati Patil and C P Kaviraj Plant Tissue Culture Laboratory, Department of Botany, Gulbarga University, Gulbarga 585 106, India Received 8 March 2004, revised 25 November 2004, accepted 15 December 2004 Effect of different growth regulators and their concentration on callus formation and organogenesis in various explants and callus was studied in mung bean [Vigna radiata (L.) Wilczek. 2,4-D and NAA alone or in combination with Kn supported callus induction and further growth. 2,4-D proved better than NAA and addition of Kn at 4.6 (μM/L) further enhanced the growth of callus. Organogenesis was obtained from callus, shoot tip and cotyledonary node explants on BAP medium. Keywords: Vigna radiata, callus induction, micropropagation, mung bean IPC code: Int. Cl.7 A01H4/00, 5/04, 5/06

Introduction Legumes have high nutritional value; hence they have been subjected to many studies for potential improvement through cell and tissue culture1-4. Although they are difficult to manipulate in vitro, some progress has been made in recent years5-9. Vigna radiata (L.) Wilczek is considered as an important pulse crop. Attempts made for further improvement of this crop by conventional breeding methods met with limited success10. Earlier studies on V. radiata have been carried out on its two cultivars, M2-5 and S-8 for the regeneration of seed on basal MS medium11, and also reported that only cytokinin (BA) was used for shoot regeneration from 2-day-old cotyledons. Cotyledons of these two cultivars, as a source of explant, were used only for regeneration and not for callus induction12. In the present study, callus induction and organogenesis from different explants, viz. cotyledon, cotyledonary nodes, shoot tip and callus has been reported in V. radiata cv PS-16.

sterilized with 0.1% mercuric chloride (W/V) for 3 min, washed thoroughly with sterile distilled water to remove the traces of mercuric chloride. Seeds were germinated on double layers of pre-sterilized moistened filter paper in culture tubes (25 × 150 mm) and incubated at 26±2°C under a light intensity of 3000 lux provided by cool white fluorescent lamps. Germinated seeds of 3-4 days were used for the isolation of cotyledons, cotyledonary nodes and shoot tip. Explants of cotyledons, cotyledonary nodes measuring about 1.0 cm in length and shoot tip measuring about 3-4 mm in length were isolated in laminar air flow cabinet and implanted on MS and B5 media13,14 supplemented with different concentrations of 2,4-D (2,4-Dichloro phenoxy acetic acid), NAA (α-Naphthalene acetic acid), Kn (Kinetin) and BAP (6-Benzylaminopurine) containing 3% of sucrose and 0.8% agar. All the media were adjusted to pH 5.8 before autoclaving. Statistical Analysis

Materials and Methods Seeds of V. radiata cv PS-16 were obtained from Agricultural Research Station, Gulbarga. They were dipped in 70% ethanol for 3 min and then surface

Twenty-five cultures were raised at each treatment and all the experiments were repeated thrice. Data taken after 30 days of culture were subjected to students t-test.

_________________ *Author for correspondence: Tel: 91-8472-245731; Fax: 91-8472-245297 E-mail: [email protected]

Results and Discussion Morphogenetic potentiality of cotyledons and cotyledonary node and shoot tip explants from 3-5-

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day-old seedlings were studied. It was possible to induce callus successfully from all the explants used on MS and B5 media, supplemented with 9.05 (μM/L) 2,4-D. Lower concentrations of 2,4-D (2.26 and 4.52 μM/L) did not favour callus induction. MS medium proved slightly better than B5 medium. Callus was initiated from hypocotyl within 3-4 days followed by cotyledons in 5-6 days and cotyledonary node in 6-8 days. Frequency of callus induction was studied on both MS and B5 media. In hypocotyl explants, 100% callus induction was noticed on both MS and B5 media followed by cotyledons and cotyledonary nodes (Table1, Fig. 1). The difference in the time required for callus initiation and frequency of callus induction amongst various explants has been reported in other legumes like Cicer arietinum15,16 and Cajanus cajan7,8,17. In the present study, hypocotyl was also found to be a better source for callus induction, as reported earlier in Sesbania grandiflora18, Arachis hypogea19 and V. radiata20. Effect of Synthetic Auxins on Callus Growth

Since MS medium gave good results than B5 medium, it was further used to study the effect of two synthetic auxins viz; 2,4-D and NAA on the growth of callus. Lower concentration (2.26 to 4.52 μM/L) of 2,4-D and NAA poorly support the growth of callus, whereas, 9.05 μM/L 2,4-D and 10.72 μM/L NAA supported the growth of callus. All the concentrations of 2,4-D proved better than NAA and the results were statistically significant. However, higher concentrations of 2,4-D (13.57 μM/L) and NAA (16.05 μM/L) reduced the fresh and dry weight of callus (Table 2).

Fig. 1⎯Callus initiation and multiple shoot from various explants in V. radiata on MS medium with 9.05 μM/L 2, 4-D and 8.90 μM/L BAP: (a) Callus initiation from cotyledon explant; (b) Callus initiation hypocotyl explant; (c) Callus initiation from cotyledonary leaf explant; (d) Initiation of multiple shoots from cotyledonary nodal explants; (e) Initiation of multiple shoots from cotyledon explant; (f) Multiple shoots from shoot tip along with callus at the base. Table1⎯Frequency of callus induction from different explants of V. radiata on medium containing 9.05 μM/L 2,4-D Source of explant

Effect of Supplementing Kn on Callus Growth

Kn was supplemented in the range of 2.329.30 μM/L along with 9.05 μM/L 2,4-D. The Kn concentrations of 2.32 μM/L enhanced the fresh and dry weight of callus. However, 9.30 μM/L concentration of Kn reduced the growth of callus. Maximum amount of callus was recorded on MS medium supplemented with 9.05 2,4-D + 4.60 μM/L Kn as compared to other concentrations tested, which proved statistically significant (Table 3, Fig. 1). Regeneration from Shoot Tips

Shoot tips, about 3-4 mm in length, were inoculated on MS basal medium free from growth regulators and with 2.22 μM/L BAP, resulted in the formation of single plantlets in 76.5 and 68.5% of the

Hypocotyl Cotyledon Cotyledonary node

Frequency of callus induction (%) MS B5 100 80.2 72.5

100 74.6 62.0

±=Standard error Data based on 25 explants per treatment *=Significant at 5% level **=Significant at 1% level

cultures, respectively. On 4.4 and 8.9 μM/L BAP supplemented media, 3.5 and 4.6 average number of shoots per explant developed in 70.6 and 80.9% cultures, respectively. Higher concentrations of BAP (13.30 & 22.20 μM/L) reduced the frequency of shoot formation to 70.2 and 60.6%, respectively, but increased the number of shoots formation per culture

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5.6 and 5.8, respectively. Compared to all the concentrations of BAP, the maximum shoot length was recorded in 8.90 μM/L (Table 4, Fig. 1), which was statistically significant. Regeneration from shoot tips has been earlier reported in V. radiata11,21. Regeneration from Cotyledons

The response of cotyledonary explants after the removal of embryo axis on MS and B5 media alone or supplemented with various concentrations the BAP. Table 2⎯Effect of 2,4-D and NAA on hypocotyl derived callus of V. radiata on MS medium (30 days after growth) Conc. of synthetic auxins (μM/L) 4.52 9.05 13.57 5.36 10.72 16.05

Growth of the callus (mg) 2,4-D NAA FW DW FW DW 641±5.5* 872±2.3** 540±2.6 00.00 00.00 00.00

62.4±2.0* 89.5±1.8** 56.5±1.8 00.00 00.00 00.00

00.00 00.00 00.00 00.00 00.00 00.00 452±2.5* 43.5±3.0* 676±3.2** 65.4±2.8** 342±2.6 30.6±1.8

Data based on 25 explants per treatment ±=Standard error * Significant at 5% level **=Significant at 1% level

Addition of BAP to MS basal medium induced slight callus formation followed by shoot differentiation, on both MS and B5 medium. Shoot buds were not observed on MS basal media, but addition of BAP at 2.22 μM/L produced green shoot bud like structures. BAP induced healthy multiple shoots at 8.9 μM/L. The frequency and number of shoots per explant increased on medium supplemented with BAP at 13.30 and 22.20 μM/L, however, length of the shoots decreased in these treatments (Table 4; Fig. 1). Regeneration from Cotyledonary Nodal Segments

Cotyledonary nodal segments were excised from 56-day-old seedlings and cultured on MS and B5 media supplemented with 4.4-22.20 μM/L BAP. Shoots were produced from the nodal region in all the concentrations of BAP. The frequency of shoot induction was 100% at 8.90 μM/L BAP and decreased to 80 and 50%, respectively at 13.30 and 22.20 μM/L BAP. The maximum number of shoots was found on the medium containing 13.30 μM/L BAP as compared to other concentrations (Table 4; Fig. 1). IAA concentrations at 1.42 and 2.85 μM/L along with BAP (8.90 μM/L) showed increase in the number of shoots per culture and also the shoot length.

Table 3⎯Effect of supplementing kinetin along with 9.05 μM/L 2,4-D on the growth of hypocotyl derived callus in V. radiata on MS medium 2,4-D + Kn (μM/L)

FW

10 days DW

FW

20 days DW

FW

30 days DW

9.05 + 0.00 9.05 + 2.32 9.05 + 4.60 9.05 + 6.92

360±3.0 480±6.0* 598±5.0** 326±4.0

32.6±1.2 42.6±1.4* 55.4±1.3** 30.6±1.8

660±5.0 752±6.0* 856±4.0** 436.0±4.0

62.6±1.2 72.6±1.3* 84.2±1.6** 40.6±1.7

870±4.0 1020±6.0* 1280±8.0** 556±6.0

82.6±1.2 98.2±1.0* 110.3±1.3** 52.5±1.4

Data based on 25 explants per treatment ± = Standard error * = Significant at 5% level ** = Significant at 1% level Table 4⎯Morphogenetic response of cotyledons, shoot tips and cotyledonary node explants of V. radiata on MS medium supplement with different concentrations of BAP Concentration Frequency (%) Cotyledonary (μM/L) Cotyledon Shoot tip node 4.40 8.90 13.40 22.20

45±1.3 70.6±1.3* 50±0.8* 80.9±1.3** 60±1.3** 70.2±1.4* 72±1.0** 60.6±1.2

Data based on average of 25 explants ± = Standard error * = Significant at 5% level ** = Significant at 1% level

100±0.0** 100±0.0** 80±0.0* 50±0.0

No. of shoots per culture Shoot length per culture Cotyledon Shoot tip Cotyledonary Cotyledon Shoot tip Cotyledonary node node 1.0±0.0 2.5±1.2* 3.0±0.6* 3.5±0.7**

3.5±0.8 4.6±0.6 5.6±0.8* 5.8±0.6**

2.0±0.8 2.5±0.8* 3.2±0.9** 2.6±0.8*

1.2±0.8 1.5±0.4* 2.0±0.3** 1.5±0.2*

1.5±0.7* 2.0±0.4** 1.5±0.6* 1.5±0.3*

1.5±0.8* 3.6±0.6** 1.2±0.4 1.2±0.4

RAO et al: CALLUS INDUCTION AND ORAGANOGENESIS IN V. RADIATA

Regeneration from Callus

Thirty-day-old callus derived from hypocotyl was transformed to regeneration media supplemented with different concentrations (2.22 to 8.90 μM/L) of BAP. The 2.22 μM/L BAP did not support the formation of multiple shoots. The BAP at 4.40 and 8.90 μM/L favoured shoot formation within 20 days of culture with low frequency. The frequency of shoot formation was 20.6% at 4.40 μM/L BAP and increased to 26.8% at 8.90 μM/L BAP. About 3.6 to 4.2 average number of shoots was formed per culture. The addition of NAA at 1.34 and 2.68 μM/L further enhanced the frequency of shoot formation. The average number of shoots per culture also increased to 6.2 and 6.8, respectively (Table 5, Fig. 2). Rooting of Regenerated Plants

Well developed shoots, about 2-3 cm in length, were excised and transferred to 1/2 strength MS medium supplemented with various concentrations of NAA and IBA. IBA proved to be more helpful than NAA in root formation. The frequency of root formation and the number of roots per culture increased with the increase in IBA concentration from

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1.23 to 4.90 μM/L IBA. The maximum number of roots formed was on IBA (4.90 μM/L) supplemented medium (Table 6, Fig. 2), which was significant statistically. Hardening and Field Establishment of Plantlets

The regenerated plantlets were gently separated from the adherent agar and transferred individually to 1/2 strength MS medium. In this medium, the roots attained a length of about 2-3 cm and became stouter within a week. The plantlets were then transferred to polythene cups containing autoclaved sand and soil mixture (2:1). Initially the pots were covered with perforated polythene bags for one week to prevent desiccation and to maintain humidity. Of the plantlets transferred to fields only 10% healthy plants survived, and those which were very feeble could not stand erect unless the support. Efforts were made to increase the survival percentage of plants in the field and to obtain stronger plants (Fig. 2). From the results, it is clear that induction of callus depends upon the type of explants and growth regulators. Hypocotyl explants proved to be excellent in terms of time taken, frequency and growth of

Table 5⎯Multiple shoot induction from hypocotyl derived callus on MS medium supplemented with various concentrations of BAP alone or in combination with NAA Growth Regulators (μM/L)

Frequency of shoots inductions (%)

No. of shoots/explant

Height of plantlets (cm)

0.0±0.0 20.6±1.6 26.8±2.4 30.6±3.2* 40.2±2.8**

0.0±0.0 3.6±0.8 4.2±0.6 6.2±0.8* 6.8±0.8**

0.0±0.0 2.5±0.2 2.8±0.8 3.4±0.8* 5.2±0.9**

BAP (2.22) BAP (4.40) BAP (8.90) BAP(8.90)+NAA(1.34) BAP(8.90)+NAA(2.68) Data based on average of 25 explants ± = Standard error * = Significant at 5% level ** = Significant at 1% level

Table 6⎯Induction and frequency of rooting of the plantlets in different concentration of auxins supplemented to MS medium Concentration of auxins (μM/L)

Frequency of root induction (%)

No. of roots per explant

Response

30.8±0.8 40.9±0.4** 35.2±0.3* 30.6±0.7

3.6±0.8 4.0±0.2 5.2±0.3* 6.8±0.8**

Very thin roots Thick roots Thick roots Thick roots with hairy like sec. roots

MS +(0.00) MS +(2.46) IBA MS + (2.68) NAA MS +(4.90) IBA Data based on average of 25 explants ± = Standard error * = Significant at 5% level ** = Significant at 1% level

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Fig. 2⎯ (a) Multiple shoots from callus on MS + 8.90 μM/L BAP; (b) Root induction on MS medium supplemented with 2.46 μM/L IBA. Hardened plant in polycups containing sterile soil + vermiculate (2:1).

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