EFFECTIVE CALLUS INDUCTION AND PLANT REGENERATION IN BRASSICA NAPUS (L.) VAR DGS-1

Journal of Cell and Tissue Research Vol. 12(2) 3229-3234 (2012) ISSN: 0974-0910 (Available online at www.tcrjournals.com) Original Article EFFECTIVE CALLUS INDUCTION AND PLANT REGENERATION IN BRASSICA NAPUS (L.) VAR DGS-1 SHARMA, M. AND GUPTA, S. K. Department of Plant Breeding and Genetics, SKUAST-J, Chatha, Jammu (J&K) 180 009. E. mail: munishsangra@gmail.com Received: May 31, 2012; Accepted: July 6, 2012 Abstract: The establishment of cultures was done by inoculating the stem sections measuring 0.5-1.0 cm of Brassica napus var. DGS-1 in vitro grown seedlings on modified MS medium supplemented with various concentrations and combinations of different growth regulators viz., 2,4-D, BAP and Kinetin. Response to callus induction differed accordingly to the levels of hormonal treatments. Among various Auxins used, 2, 4-D produced the best results with respect to callus formation. Fully developed calli were formed in the media containing 2, 4 D (1.5 mg l -1 ). In this combination, friable calli were formed which were initially yellowish green in colour and later turned pale yellow. Callus induction was lower at other levels of 2, 4 D. However, increasing 2, 4-D beyond 1.5mg l -1 suppressed callus induction. For shoot regeneration, the calli were cut into small pieces and cultured on MS medium supplemented with different combinations and concentrations of various growth regulators viz, BAP, NAA, Kn, IAA and AgNO 3. The maximum shoot regeneration was observed on the medium containing 3 mg l -1 Kn, 0.50 mg l -1 NAA and 1 mg l -1 AgNO 3. In vitro grown shoots were separated and transferred to different combinations of rooting media i.e. MS medium supplemented with various concentrations of two Auxins viz. NAA and IAA. The maximum root regeneration was observed on the media containing MS half strength (hormone free) + 15 g l -1 sucrose. Key words: Brassica napus, Hormonal treatment, Callus induction? INTRODUCTION In non-conventional improvement of crop plants, the establishment of an efficient plant culture technique represents a basic step. Brassica napus is considered as the most important source of vegetable oil and protein-rich meal worldwide [1]. In India, Brassica napus var. Gobhi Sarson is slowly replacing Raya in irrigated areas [2]. The crop is gaining importance because of its high yield potential of 20q/ha [3], wide adaptability, suitability for late planting and a high oil content (44.6%) of good quality [4]. The productivity in Brassica napus is affected by a number of biotic and abiotic factors. In order to cope up with these factors and to increase the productivity, conventional breeding approaches have to be supplemented by nonconventional approaches. Biotechnological techniques such as genetic engineering require reliable and efficient tissue culture protocol. This area needs to be researched for a high frequency regenerative system because it is a preliminary step towards advanced non-conventional techniques such as genetic transformation [5,6]. The present investigation reports the standardized protocol for callus induction, shoot and root regeneration in Brassica napus L. var. DGS-1. MATERIALS AND METHODS Plant material: The plant materials consisted of certified seeds of Brassica napus var. DGS-1, which were procured from the Division of Plant Breeding and Genetics, Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu. The seeds were raised under in vitro conditions by inoculating them on Murashige and Skoog [7] full 3229

J. Cell Tissue Research strength basal medium containing 3 per cent sucrose with ph 5.8. Twenty days old seedlings were used as the source of the explants from which stem sections measuring 0.5-1.0 cm were used for establishment of callus cultures. Establishment of callus cultures: The establishment of cultures was done by inoculating the stem sections of in vitro grown seedlings on modified MS medium supplemented with various combinations of different growth regulators viz., 2,4- D, BAP and Kinetin (Table 1). The cultures were then incubated for callus initiation and the days taken for proliferation were recorded. Callus multiplication: The growing callus from the established cultures was separated from the initial explants and sub cultured in fresh medium. Visual observations with respect to growth, type and colour of the callus were recorded. On the basis of these observations, the callus was again sub-cultured onto the medium giving the best results for further multiplication. Organogenesis and shoot multiplication: To optimize the culture medium for shoot regeneration, the calli were divided into small pieces and cultured on modified MS medium supplemented with various combinations and concentrations of growth regulators viz., BAP, NAA, Kn, IAA, and AgNO 3 (Tables 2,3). For every combination, 8 flasks with 5 pieces of calli were inoculated. The regenerated shoots were then multiplied on the medium showing best shoot multiplication. Rooting of the plants: After a desirable rate of multiplication was achieved, the multiplied shoots were separated and individual shoots were transferred onto MS medium with or without auxins (NAA and IAA). Ten replicates for each treatment were used. The medium on which best root growth was observed was used for subsequent rooting experiment (Table 4). RESULTS For germination, seeds were cultured on both half strength and full strength MS medium (Murashige and Skoog, [7], supplemented with 3 per cent sucrose. However, half strength MS medium was found to be better as compared to full strength medium because on this medium seed germination started within 4 th day of culture while on the full strength MS medium, it could be initiated only after 7 days. However, per cent of germination was same (97%) in both the media. Establishment of callus cultures: The stem sections obtained from the in vitro germinated seedlings of DGS-1, were cut into small pieces of about 0.5 1.0 cm in size and subsequently cultured on MS medium supplemented with various concentrations and combinations of different growth regulators viz., 2, 4-D, BAP and kinetin and the growth response of these explants on different media including the changes in colour and texture of calli, were recorded. A total of 15 combinations were tried (Table 5). Among various Auxins used, 2, 4-D produced the best results with respect to callus formation. Fully developed calli were formed in the media containing 2, 4 D (1.5 mg l -1 ). In this combination, friable calli were formed which were initially yellowish green in colour and later turned pale yellow. Callus induction was lower at other levels of 2, 4 D. However, increasing 2, 4-D beyond 1.5mg l -1 suppressed callus induction. Calli which were pale yellow and friable in texture were maintained by transferring them on the same but fresh medium every three weeks and hence this medium was used for the induction of callus in the subsequent experiments. In this medium, callus induction took place within 4-5 days. However, the growth of callus was slow at the initial stage. Therefore, first sub-culturing was done after 6 weeks, followed by subsequent sub-culturing at an interval of three weeks. After 2-3 sub-cultures, calli were transferred to maintenance medium (MS basal medium supplemented with 0.2 mg l -1 of 2, 4 D). Shoot regeneration: For shoot regeneration, the calli were cut into small pieces and cultured on MS medium supplemented with different combinations and concentrations of various growth regulators viz, BAP, NAA, Kn, IAA and AgNO 3. Out of 56 media tried, the best results were obtained in MS medium supplemented with 3 mg l -1 Kinetin, 0.5 mg l -1 NAA and 1mg l -1 AgNO 3. In this case, the calli turned green in just 9 days and shoot proliferation initiated after 14 days (Tables 6,7). Shoots of 2-5 cm length were formed in this combination after 26 days. These shoots were separated and cultured on the same medium for further multiplication. The numbers of shoots per explant increased further after subsequent sub-culturing and maximum of 5 shoots were 3230

Sharma and Gupta Table 1: Different concentrations and combinations of growth regulator for the establishment of callus cultures CIM denotes Callus Initiation Medium Table 2: Different concentrations and combinations of growth regulators used for shoot regeneration. SRM denotes Shoot Regeneration Medium S. No. S. No Medium Medium composition (MS supplemented with) 1. CIM 1 0.5 mg l -1 2,4-D + 1.0 mg l -1 BAP 2. CIM 2 1.0 mg l -1 2,4-D +1.5 mg l -1 BAP 3. CIM 3 1.5 mg l -1 2,4-D + 2.0 mg l -1 BAP 4. CIM 4 2.0 mg l -1 2,4-D + 2.5 mg l -1 BAP 5. CIM 5 2.0 mg l -1 2,4-D + 3.0 mg l -1 BAP 6. CIM 6 0.5 mg l -1 2,4-D + 1.0 mg l -1 Kn 7. CIM 7 1.0 mg l -1 2,4-D + 1.5 mg l -1 Kn 8. CIM 8 1.5 mg l -1 2,4-D + 2.0 mg l -1 Kn 9. CIM 9 2.0 mg l -1 2,4-D + 2.5 mg l -1 Kn 10. CIM 10 2.0 mg l -1 2,4-D + 3.0 mg l -1 Kn 11. CIM 11 0.5 mg l -1 2,4-D 12. CIM 12 1.0 mg l -1 2,4-D 13. CIM 13 1.5 mg l -1 2,4-D 14. CIM 14 2.0 mg l -1 2,4-D 15. CIM 15 2.5 mg l -1 2,4-D Amount of cytokinin (mg l -1 ) + Auxin (mg l -1 Name of media Kn + IAA Kn + NAA BAP + IAA BAP + NAA 1. 2 + 0.25 I SRM1 II SRM1 III SRM1 IV SRM1 2. 3 + 0.25 I SRM2 II SRM2 III SRM2 IV SRM2 3. 4 + 0.25 I SRM3 II SRM3 III SRM3 IV SRM3 4. 5 + 0.25 I SRM4 II SRM4 III SRM4 IV SRM4 5. 2 + 0.50 I SRM5 II SRM5 III SRM5 IV SRM5 6. 3 + 0.50 I SRM6 II SRM6 III SRM6 IV SRM6 7. 4 + 0.50 I SRM7 II SRM7 III SRM7 IV SRM7 8. 5 + 0.50 I SRM8 II SRM8 III SRM8 IV SRM8 9. 2 + 0.75 I SRM9 II SRM9 III SRM9 IV SRM9 10. 3 + 0.75 I SRM10 II SRM10 III SRM10 IV SRM10 11. 4 + 0.75 I SRM11 II SRM11 III SRM11 IV SRM11 12. 5 + 0.75 I SRM12 II SRM12 III SRM12 IV SRM12 produced in 4 th sub culture. For every combination, 8 flasks with 5 pieces of calli were inoculated. Shoot multiplication: The individual shoots from the shoot clumps were separated by splicing with sharp knife and cultured on with 3 mg l -1 Kinetin, 0.5 mg l -1 NAA and 1mg l -1 AgNO 3 combination of culture medium till sufficient rate of multiplication was achieved. After sub-culturing for 36 days, it was observed that the rate of shoot multiplication increased considerably. Root regeneration: In vitro grown shoots were separated and transferred to different combinations of rooting media i.e. MS medium supplemented with various concentrations of two Auxins viz. NAA and IAA and observations were recorded periodically. In all 8 combinations were tried. After 30 days of Table 3: Modified media composition for shoot regeneration S.No. Amount of cytokinin (mgl -1 ) + Kn+NAA+AgNO 3 Auxin (mgl -1 )+AgNO 3 1. 3+0.25+1.0 II SRM2(1) 2. 3.0+0.25+2.0 II SRM2(2) 3. 4.0+0.25+1.0 II SRM3(1) 4. 4.0+0.25+2.0 II SRM3(2) 5. 3+0.50+1.0 II SRM6(1) 6. 3+0.50+2.0 II SRM6(1) 7. 4+0.50+1.0 II SRM7(1) 8. 4+0.50+2.0 II SRM7(2) Table 4: Different concentrations of growth regulators used for root induction. RIM denotes root initiation medium. S. No Medium Medium composition 1. RIM 1 MS full strength + 0.05 mg l -1 IAA + 30 g l -1 Sucrose 2. RIM 2 MS full strength + 0.10 mg l -1 IAA + 30 g l -1 Sucrose 3. RIM 3 MS full strength + 0.20 mg l -1 IAA + 30 g l -1 Sucrose 4. RIM 4 MS full strength + 0.05 mg l -1 NAA + 30 g l -1 Sucrose 5. RIM 5 MS full strength + 0.10 mg l -1 NAA + 30 g l -1 Sucrose 6. RIM 6 MS full strength + 0.20 mg l -1 NAA + 30 g l -1 Sucrose 7. RIM 7 MS half strength (hormone free) + 15 g l -1 Sucrose 8. RIM 8 MS half strength (hormone free) + 30 g l -1 Sucrose culture in half strength MS (hormone free) and 15g l - 1 sucrose, 4-6 roots appeared per explant with an average length of 6 cm (Table 8). Time duration for appearance of the roots: This media supported as the best medium for root growth. Hundred per cent rooting was observed, roots started appearing after 10-15 days. Plants were ready for transfer to pots within 25 days. DISCUSSION Regeneration in Brassica napus is highly variable and genotype specific. Several papers have reported regeneration of shoots from seedlings or mature plant derived explants of B. napus [8]. To date organogenesis has been achieved in a variety of explants such as stem sections [9], stem thin-cell layer [10], leaf discs [8], roots [11], cotyledons [12 and hypocotyls [13]. In this experiment, the stem sections measuring 0.5-1.0 cm from in vitro grown seedlings were incubated for callus initiation and the days taken for proliferation were recorded. Fifteen combinations were tried, but friable and fully developed calli were formed in the media containing 2, 4-D (1.5 mg l -1 ). These findings are in conformity with many earlier reports wherein 2, 4-D was observed to be an essential component of callus induction media. A similar concentration (1.5 3231

J. Cell Tissue Research Fig. 1: Callus induction and plant regeneration in Brassica Napus (L.) var. DGS-1 A B A,B: Calli formation in Brassica napus C C: Multiple shot regenaration from calli of Brassica napus D D: Root regeneration in Brassica napus 3232

Sharma and Gupta Table 5: Effect of different growth regulators on the establishment of callus cultures S.No. Medium Growth Response 1. CIM1 No response (Browning followed by desiccation) 2. CIM2 No response (Browning followed by desiccation) 3. CIM3 No response (Browning followed by desiccation) 4. CIM4 Moderate response but no further growth 5. CIM5 Moderate response but no further growth 6. CIM6 No response (browning followed by desiccation) 7. CIM7 No response (browning followed by desiccation) 8. CIM8 Moderate response but no further growth 9. CIM9 Moderate response but no further growth 10. CIM10 Moderate response but no further growth 11. CIM11 No response (Browning followed by desiccation) 12. Initiation of hard and compact callus but fully CIM12 developed callus was not formed 13. CIM13 Friable callus, yellowish green in colour 14. CIM14 Moderate response and callus turned brown 15. CIM15 Moderate response and callus turned brown Table 6: Effect of different combinations of growth regulators on shoot regeneration. - = No Response; + = Moderate Response; ++ = High Response S.No Medium I II III IV Kn + IAA Kn + NAA BAP + IAA BAP + NAA 1. SRM1 - - - - 2. SRM2 - + - - 3. SRM3 - + - - 4. SRM4 - - - - 5. SRM5 - - - - 6. SRM6 - ++ - - 7. SRM7 - + - - 8. SRM8 - - - - 9. SRM9 - - - - 10. SRM10 - - - - 11. SRM11 - - - - 12. SRM12 - - - - Table 7: Effect of modified media composition for shoot regeneration. + = Moderate Response; ++ = High Response S.No. Medium Response 1. II SRM2(1) + 2. II SRM2(2)) + 3. II SRM3(1) + 4. II SRM3(2) + 5. II SRM6(1) ++ 6. II SRM6(2) + 7. II SRM7(1) + 8. II SRM7(8) + Table 8: Effect of different concentrations of growth regulators on root induction. - = no responce; + = moderate responce;++ =high responce. S.No. Medium Response 1. RIM 1-2. RIM 2-3. RIM 3-4. RIM 4-5. RIM 5-6. RIM 6 + 7. RIM 7 ++ 8. RIM 8 - mg l -1 ) of 2, 4-D was also found to be adequate for callus induction from hypocotyl segments of B. napus on B 5 medium [14]. Vyvadilova and Zelenkova [15] also observed that best callus initiation form axillary and terminal meristems, leaves, hypocotyls and roots was in MS media with 0.5-2.0 mg l -1 2,4 D. The maximum shoot regeneration was observed on the medium containing 3 mg l -1 Kn, 0.50 mg l -1 NAA and 1 mg l -1 AgNO 3. Present results are in conformity with the earlier reports by Singh and Chandra [16] and Singh [17] who reported that high concentrations of two cytokinins viz; BAP and kinetin is required for induction of shoot from cotyledons, leaf lamina and leaf mid rib explants. Williams et al. [18] has also suggested incorporation of AgNO 3 (1-10 mg l -1 ) into the culture medium of Brassica oleracea var. gemmifera callus which significantly improved its growth and allowed long term callus culture. In the absence of AgNO 3, the callus died shortly after removal from the hypocotyl explants. Regeneration of shoots from callus on low hormone medium was also enhanced by AgNO 3. Therefore, on the basis of the results obtained, it is confirmed that in order to produce maximum number of shoots per explant, a high cytokinin/auxin ratio should be maintained, which in the present case was found to be 6 : 1 as the best results for organogenesis were obtained at this ratio. In case of root induction, effects of with or without different concentrations of growth regulators viz., IAA and NAA were studied. Out of 8 media tried, the best media for root induction was MS half strength (hormone free) + 15 g l -1 sucrose. These results are in confirmation with the earlier report of Ono et al. [19] who reported that 2.0 mg l -1 NAA was effective for root formation but was qualitatively poorer. This suggests that an increase in the concentration of NAA leads to increased rooting up to 2.0 mg l -1 NAA. Yun et al. [20] have shown maximum number and length of root induction in Brassica napus on half strength MS medium without any growth regulator. On the basis of the results obtained, it is confirmed that in order to produce maximum number and length of roots, the shoots are to be inoculated on MS medium half strength without any growth regulator. Abbreviations used: 2, 4-D (2, 4-dichlorophenoxyacetic acid), BAP (Benzyl amino purine), NAA 3233

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