www.wjpr.net Vol 4, Issue 1, 2015. 644
CALLUS INDUCTION AND ORGANOGENESIS IN
CAPSICUM
ANNUUM L. CV PUSAJWALA AND G4
Srinath Rao*# and Prathiba Sangapure
Plant Tissue Culture and Genetic Engineering Laboratory, Department of Post-Graduate Studies and Research in Botany, Gulbarga University, Gulbarga - 585 106, Karnataka, India
#Presently Registrar(Evaluation) Karnataka State Women’s University Bijapur
ABSTRACT
Callus induction and plant regeneration in chilli pepper (Capsicum
annuum L.) cv pusajwala and G4 was investigated. Cotyledon and hypocotyl explants excised from 10-12 days old seedlings were cultured on Murashige & Skoog’s (MS) medium supplemented with different concentrations of growth regulators like 2, 4-D, NAA, BAP, and Zeatin. Both the explants were found suitable for induction of callus on MS medium supplemented with NAA at 2.0mg/l. Hypocotyl was found to be a better explant for the induction of maximum callus. Healthy multiple shoots were obtained from the cut ends of cotyledonary leaf segments for both the varieties on MS containing 1.0 mg/l BAP and 0.1 mg/l IAA. The maximum root induction from the regenerated shoots was achieved on half the strength MS medium supplemented with 0.2 mg/l IAA. The in vitro
grown plantlets were successfully transplanted into soil where they flowered and produced fruits identical to those developed by control plants.
KEY WORDS: Chilli, callus, multiple shoots, 2, 4-D, BAP
INTRODUCTION
Chili pepper (Capsicum annuum L.) is one of the most important vegetable cum spice crop around the world and account for about 22 % of total global spices trade composition. Chillies contain numerous chemicals such as volatile oil, fatty oils, capsaicinoids, carotenoids, vitamins, protein, fiber and mineral elements. [1] The chili fruit powder is the most important savory ingredient in the Indian traditional dishes. Carotenoids present in chilli
Volume 4, Issue 01, 644-657. Research Article ISSN 2277– 7105
Article Received on 26 October 2014, Revised on 16 Nov 2014, Accepted on 08 Dec 2014
*Correspondence for
Author
Srinath Rao
Presently
www.wjpr.net Vol 4, Issue 1, 2015. 645 extracts were found to have a synergistic anti-mutagenic and in vitro anti-tumor-promoting activity. [2, 3] However, it is highly susceptible to many pathogens like viruses, bacteria, fungi and insect-pests. [4] Propagation of plants through tissue culture offers a unique advantage over conventional breeding methods in production of disease-free plants and release of improved cultivars, Approaches are being made to produce genetically engineered disease resistance pepper varieties, but they have been delayed due to its recalcitrant nature. [5, 6]
Capsicum has been categorized as one of the most recalcitrant plant species to manipulate in tissue culture due to the formation of ill-defined buds or shoot like structures either resisting elongation or producing rosettes of distorted leaves which generally do not produce normal shoots. [5] In Capsicum, several procedures are available for inducing in vitro plant regeneration. [7-9] For gene transfer technology to be useful, however, in vitro regeneration of complete and fertile plants is required
MATERIAL AND METHODS
Seeds were thoroughly washed under running tap water and then with distilled water. After a 1minute treatment with 70% ethanol the seeds were surface sterilized with 0.1% Mercuric Chloride (HgCl2 ) solution for about 5 minutes followed by three washes with sterilized distilled water to remove the traces of HgCl2 solution. They were inoculated on filter paper bridges dipped in sterile distilled water in culture tubes (25 × 150 mm). Hypocoty and cotyledons from10-12 days old seedlings were used as explants. Explants about 5-6 mm in length, were inoculated in such a way that lower surface of cotyledons and cut end of hypocotyls was in contact with the media, each culture tube contained one explant. All cultures were incubated in culture room at 25± 20C temperature and an automatic electronic timer was used to regulate the required photoperiod. Throughout the culture period, 16 hours light and 8 hours dark period was maintained, provided by cool white fluorescent light intensity of 3000 lux. For induction of callus, MS basal medium [10] supplemented with different kinds of auxins (1-3mg/l concentration) either alone or in combination with Kinetin. (0.5-2.0mg/l) were standardized.
www.wjpr.net Vol 4, Issue 1, 2015. 646 For induction of multiple shoots MS medium supplemented with different types of cytokinins 6-benzyl amino purine (BAP), Kinetin (Kn) and Thidiazuron (TDZ) either alone or in combination with auxins was used. After 30 days, in vitro propagated shoots of 4-5 cm long, with at least 3-4 inter nodes were excised from shoot clumps and transferred to MS medium containing different concentrations (1 to 3mg/l) of Naphthalene Acetic acid (NAA) or Indole Butyric Acid (IBA).
After obtaining sufficient rooting on rooting medium, the plantlets were removed carefully and washed with distilled water to remove any adherent media then were transferred to small poly cups containing sterilized soil and sand in the ratio of 3:1 respectively. Poly cups were kept in culture room during day and were kept outside during night for about two weeks. Moisture content was maintained by spraying distilled water, 2-3 times in a day. Then these plants were acclimatized to natural condition.
RESULTS
Number of days required for callus initiation in more than 50% of explants and frequency of callus induction was determined on MS medium supplemented with 2mg/12,4- D + 1mg of Kn and the data is presented in Table-1. From the data it can be noticed that irrespective of cultivar used callus was initiated within 5-6 days from cotyledonary explants and 8-10 days from hypocotyls explants. The frequency of callus induction was highest in cotyledonary explants (80%) and it was (60%) in hypocotyl explants (Plate 1a&b).
4-www.wjpr.net Vol 4, Issue 1, 2015. 647 D or NAA as source of auxins. NAA proved to be less effective for callus induction at any of the concentration tested when compared to 2, 4-D. Since 2, 4-D at 2mg/l supported better growth of callus, further studies were carried out to investigate the interaction between 2, 4-D and kinetin at concentrations varying between 0.5 to 1.5 mg/l. From the data presented in Table-3, it can be noticed that increasing the concentration of kinetin from 0.5 to 1mg/l a gradual increase in the growth of callus was noticed (Plate-I c &d)) however Kn., at 1.5mg/l inhibited the growth of callus. Maximum callus was noticed on MS medium supplemented with 2mg/l 2, 4-D+1mg/l Kn (745.6± 0.54 mg).
Differentiation of adventitious buds was noticed from cotyledonary, hypocotyls and callus explants of both the cultivars Pusa jwala and G4 on MS medium supplemented with cytokinins. However, the time required for shoot bud initiation number of shoots / explants differed considerably depending upon the cultivars, concentration and type of cytokinins (BAP, Kn. And TDZ) used alone or in combination with IAA. Cotyledonary explant responded earlier (10-15 days) for induction of shoot buds than hypocotyl explant (14-18 days). The frequency of multiple shoot induction was highest (70%) in cotyledonary explants followed by hypocotyl explants (50%). numbers of shoot buds per explants were more in cotyledonary explant than in hypocotyls explants. Pusa jwala responded better than G4. The response of cotyledons for multiple shoot induction was better than hypocotyls. (Plate-II a&b).
www.wjpr.net Vol 4, Issue 1, 2015. 648 cytokinins and IAA, didn’t show much elongation most of them showed leafy structure and were stunted in growth. However these shoots when transferred to medium supplemented with 1 mg/l GA3, considerable elongation of shoot was achieved (Table- Plate II c). For induction of roots different concentration of NAA and IBA (0.5 to 1.5mg/l) were used. The result are presented in the Table-7, from the data it is clear that increasing the concentrations of both the auxins, an increase in number of roots per shoot was observed, highest being on MS+1 mg/l IBA (26.8± 0.17). The roots induced on IBA supplemented medium were thick (Plate –II d), where as roots on NAA supplemented medium were thin and hair like in nature.
\
Plate: 1 Callus induction on MS Medium supplemented with 2mg/l 2, 4- D
a. Hypocotyl b. Cotyledon c & d Callus on 2mg/l 2, 4-D +1mg/l Kn. c. Cotyledon derived b. callus d. Hypocotyl derived callus (Note enhanced growth of callus in d)
Table-1: Number of days taken for callus initiation and frequency of callus induction
from different explants of Chilli (Capsicum annum) Cv. Pusa Jwala and G4 on MS medium supplemented with 2.0mg/L 2,4-D+ 1mg/L Kn.
Explants Days taken for Callus
initiation
Frequency of Callus induction
Cotyledon 5-6a 80a
Hypocotyl 8-10b 60b
a b
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Plate: II. Initiation of shoots on MS + 2mg/l BAP a. Cotyledon explants b. Hypocotyl
explant c. Callus d. Elongation of Shoots on MS + 1mg/l GA3 e. Rooting on MS + 1mg/l
IBA f. plantlet in plastic pot.
Data represents average of three replicates; each replicate consisting 25 cultures. Mean followed by the same superscript in a column is not significantly different at P=0.05 levels.
Table-2: Effect of different auxins on growth of callus derived from cotyledon and
hypocotyl explants in Cv. Pusa Jwala and G4 of Capsicum annum on MS medium.
Concentration of growth hormones (mg/L)
Pusa jwala G4
Hypocotyl (mg)
Cotyledon (mg)
Hypocotyl (mg)
Cotyledon (mg) 2,4-D
1.0 464.4±0.56b 405.6±0.25a 392.5±0.56b 342.2±0.46b
2.0 546.6±0.48a 413.6±0.79a 492.8±0.33a 409.9±0.43a
3.0 371.8±0.66c 359.8±0.14b 345.5±0.99bc 339.60.56b NAA
1.0 352.0±0.78c 299.6±0.47c 318.7±0.67c 311.5±0.54b
2.0 418.7±0.33b 328.1±0.28b 379.2±0.72b 319.6±0.47b
3.0 297.8±0.54d 276.6±0.96c 310.5±0.35c 273.1±0.68c
Data represents average of three replicates. Each replicate consisting 10 explants each. Mean ± SE.
a
a b c
www.wjpr.net Vol 4, Issue 1, 2015. 650 Mean followed by the same superscript in a column is not significantly different at P=0.05 level.
Table-3: Effect of supplementing Kinetin on average fresh weight of hypocotyl derived
callus in Cv. Pusa jwala and G4 of Capsicum annum.
Concentration of growth
hormones (mg/L) Pusa Jwala(mg/L) G4(mg/L)
2.0 mg/l 2,4-D 546.6±0.35c 414.9±0.35b
2.0 mg/l 2,4-D+0.5mg/l Kn 610.8±0.87b 447.4±0.25b
2.0 mg/l 2,4-D+1.0mg/l Kn 745.6±0.54a 579.1±0.57a
2.0 mg/l 2,4-D+1.5mg/l Kn 475.1±0.92d 389.5±0.84c
Data represents average of three replicates. Each replicate consisting 10 explants each. Mean ± SE. Mean followed by the same superscript in a column is not significantly different at P=0.05 level.
Table-4: Number of days taken for initiation of multiple shoots from cotyledonary and
hypocotyl explants of Chilli (Capsicum annum) on MS medium supplemented with
2.0mg/ l BAP.
Explants Initiation of regeneration (Days)
Pusa Jwala G4
Cotyledon 10-15a 14-18a
Hypocotyl 12-13b 14-15b
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Table-5: Frequency, number of shoots obtained from cotyledon and hypocotyl explants of Chilli (Capsicum annum) supplemented with
different concentrations of cytokinins on MS medium.
Concentrations of growth hormones
(mg/L)
Pusa jwala G4
Frequency (%)
No.of shoots /Cotyledon
Frequency (%)
No.of shoots /hypocotyl
Frequency (%)
No.of shoots /Cotyledon
Frequency (%)
No.of shoots /hypocotyl BAP
1.0 50 12.1±0.16b 48 10.6±0.27a 48 10.4±0.16b 42 9.2±0.25b
2.0 86 14.6±0.25a 56 11.3±0.41a 82 13.5±0.21a 78 12.1±0.50a
3.0 68 9.2±0.86a 32 8.3±0.14b 62 8.8±0.34c 58 6.0±0.50c
Kn
1.0 46 8.8±0.27c 38 16.9±0.14c 42 18.7±0.32c 39 8.2±0.25b
2.0 52 11.8±0.86b 48 19.0±0.14b 48 10.2±0.42b 40 11.2±0.14a
3.0 30 7.3±0.34d 30 14.8±0.27d 28 18.6±0.14c 22 5.1±0.55c
TDZ
0.25 48 2.2±0.14g 36 2.4±0.14 34 2.2±0.20e 30 00±0.0
0.50 52 5.6±0.16e 38 6.6±0.26 38 4.6±0.25d 32 00±0.0
0.75 36 3.2±0.14f 25 5.2±0.02 28 2.0±0.16e 20 00±0.0
Data represents average of three replicates. Each replicate consisting 10 explants each. Mean ± SE. Mean followed by the same superscript in a column is not significantly different at P=0.05 level.
Table-6: Effect of GA3 on in vitro shoots elongation in Chilli (Capsicum annum) on MS medium
Concntrations of growth hormones (mg/L)
Pusa jwala G4
Frequency (%) Shoot length/ culture (cm) Frequency (%) Shoot length/ culture (cm)
GA3
0.5 100 11.8±0.35b 100 10.4±0.35b
1.0 100 14.6±0.25a 100 12.3±0.25a
1.5 100 9.8±0.15c 100 8.9±0.25c
www.wjpr.net Vol 4, Issue 1, 2015. 652 Table -7: Frequency, number of roots /explant and root morphology on in vitro
obtained plantlets of Chilli (Capsicum annum) on MS medium supplemented with
different concentration of NAA and IBA.
Concentratio n of growth
hormones (mg/L)
Pusa Jwala G4
Frequenc y (%)
No. roots
/plantlets Response
Frequenc y (%)
No. roots
/plantlets Response
IBA
0.5 100 10.2±0.35c Thick
roots 100 8.9±0.88c
1 Thick roots 1.0 100 96.8±0.17a Thick
roots 100 13.1±0.45
a Thick roots 1.5 100 18.4±0.35b
Thick
roots 100
10.2±0.50 b
Thick roots NAA
0.5 100 9.8±0.85c Thin roots 100 7.4±0.35c Thin roots 1.0 100 6.7±0.64d Thin roots 100 5.8±0.68d Thin roots 1.5 100 5.8±0.35c Thin roots 100 4.7±0.75e Thin roots Data represents average of three replicates. Each replicate consisting 10 explants. Mean ± SE. Mean followed by the same superscript in a column is not significantly different at P=0.05 level.
DISCUSSION
Callus formation from cut end surfaces has been reported as a common event in several plant
[image:9.595.69.530.137.349.2]www.wjpr.net Vol 4, Issue 1, 2015. 653 The difference between the Indian and non Indian cultivars with respect to requirement of growth regulators for callus induction may be due to different genomic constitution of these cultivars. Efficient and reproducible in vitro pepper regeneration system is of most importance, as it can serve, the purpose of (a) micro propagation of special elite plants e.g., male sterile Plants of F1 plants, [6, 27] (b) Production of Transgenic plant. [28-31] The explants used in hot pepper for regenarition include shoot tip, [19, 20, 24] cotyledons, [11, 14, 17, 32] hypocotyl. [19, 20] Regeneration from both cotyledonary and hypocotyls has also been reported earlier, [20, 22, 32, 33] Commonly explants were placed on an agar solidified media supplemented with a cytokine BAP, Kinetin, Zeatin or Thidiazuron alone or along with an auxins IAA, NAA or IBA. In some rare cases spontaneous shoot regeneration from explant cultured on a medium devoid of growth regulators has been observed. [34, 35] However, in present investigation regeneration of plants was not observed on basal medium devoid of growth regulators. However in most of the papers (generally for cultivars from outside India) cited in the literature it is noticed that high BAP (5mg/l) + IAA or NAA at 1mg/l is found essential for regeneration that too with very low frequency and few number of shoots per explant. 5mg/l BAP + IAA has been reported as best combinations for regeneration. [15, 19, 20, 32, 36] In few reports it is noticed that low concentrations of BAP (2-3mg/l) is sufficient for organogenesis as reported earlier.[17, 37, 38] TDZ has been shown to induce multiple shoots in pepper. [7, 11, 33, 39, 40] The results obtained in the present investigation are in agreement with earlier reports on chili. [11, 17, 41] In all the reports it is observed that in vitro plants have to be transformed to shoot elongation medium containing IAA, NAA or GA3.
Rooting in regenerated pepper plants has been achieved on ½ MS medium devoid of any growth regulators.[21, 41] However, in the present investigations it was noticed that in vitro raised pepper plants did not show any rooting on hormone free medium. However when transferred to IBA supplemented medium rooting was observed in vitro raised plants. IBA has been shown to induce rooting in pepper by other workers. [11, 14, 15, 22, 37] There are few other reports where NAA has shown to induce rooting. [36, 40]
ACKNOWLEDGEMENT
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