VEGETOS: An International Journal of Plant ResearchOnline ISSN: 2229-4473
Print ISSN: 0970-4078

Research Article, Vegetos Vol: 30 Issue: 1

In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers

Mahitha Banala, Rajinikanth Marka, Archana Pamulaparthi and Rama Swamy Nanna*
Plant Biotechnology Research Group, Department of Biotechnology, Kakatiya University, Warangal, Telangana, India
Corresponding author : Rama Swamy Nanna
Plant Biotechnology Research Group, Department of Biotechnology, Kakatiya University, Warangal-506009(TS), India
Tel: +91-870-2567137 (R), 2461455(O); Mobile: +91-9390101665
Fax: +91-0870-2438800
E-mail: [email protected]
Received: July 27, 2016 Accepted: December 14, 2016 Published: December 19, 2016
Citation: Banala M, Marka R, Pamulaparthi A, Nanna RS (2016) In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers. Vegetos 30:1. doi: 10.5958/2229-4473.2017.00002.7

Abstract

In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers

An efficient and direct in vitro regeneration of Cajanus cajan (red gram) cv ICP 26 from cotyledon explants was established. Cotyledons of 7-8 day old in vitro grown seedlings were cultured on MS/B5 medium augmented with different concentrations (0.5-5.0 mg/L) of 6-benzylaminopurine (BAP) / Kinetin (KIN). Among these concentrations, 2.0 mg/L BAP was found to be efficient in inducing maximum number of multiple shoots with 80% of response. 80% of the elongated shoots were successfully rooted on MS medium fortified with 1.0 mg/L indole-3-butyric acid (IBA). These in vitro rooted plantlets were successfully established in soil where 80- 90% of them developed into normal and fertile plants. 20 RAPD primers were used to assess the genetic fidelity of randomly selected regenerated plantlets along with the donar plant. Out of 20 RAPD primers screened only 7 primers produced clear, distinct and scorable bands with an average of 2.5 bands per primer. All banding profiles from regenerated plantlets were monomorphic and similar to those of the mother plant. The amplification pattern confirmed the genetic uniformity of the regenerated plantlets. Thus, this reproducible regeneration protocol can be used for Agrobacterium tumefaciens mediated genetic transformation in pigeon pea....

Keywords: Cajanus cajan; Cotyledon; Direct organogenesis; Plantlet establishment; RAPD; Genetic fidelity

Keywords

Cajanus cajan; Cotyledon; Direct organogenesis; Plantlet establishment; RAPD; Genetic fidelity

Abbreviations

MS: Murashige and Skoog; BAP: N6-benzylaminopurine; Kin: Kinetin; GA3: Gibberellic Acid; IBA: Indole-3- Butyric Acid

Introduction

Pigeon pea [Cajanus cajan (L.) Millsp. ] belongs to the family Fabaceae popularly known as red gram. It is one of the major grain Legumes grown in semi-arid tropics. The crop C. cajan is widely grown for its innumerable uses. The leaf, pod and seeds are used for livestock and human nutrition and also it enhances soil fertility through biological nitrogen fixation and leaf litter [1]. However, its production and productivity are constrained by several biotic and abiotic stresses. Wild species of C. cajan provide tolerance for overcoming biotic and abiotic stresses which are not found in cultivated species, but these wild species may be associated with undesirable traits that are difficult to overcome through conventional breeding [2]. This has made it necessary to explore transgene technology for developing biotic and abiotic stress tolerant varieties. For transgene technology reproducible in vitro regeneration protocol is prerequisitive. Since legumes are recalcitrant to regenerate through tissue culture, most of the efforts have been devoted towards optimization and developing efficient in vitro regeneration system to facilitate transformation technologies [3]. In C. cajan, the plant regeneration by organogenesis has been reported through pre-existing meristems like apical meristem [4], cotyledon [5,6,7] and hypocotyl explants [4,6]. Multiple shoot induction has been achieved from cotyledonary node explants [9,10,11,12] and from epicotyl explants [6,13]. In tissue culture, genetic stability often arises as a manifestation of epigenetic influence or changes in the genome of differentiating somatic cells induced by tissue culture conditions [14,15]. Therefore, genetic stability of in vitro regenerated plantlets has always been assessed during standardization of an efficient in vitro propagation protocol. Very few reports are available describing genetic transformation of pigeon pea, probably due to lack of highly efficient and high frequency reproducible direct regeneration system which are dependent on genotype. Hence, in the present communication we report an efficient reproducible in vitro direct regeneration from cotyledon explants of Cajanus cajan cv. ICP-26. The evaluation of genetic uniformity among the regenerants.

Materials and Methods

Plant material
Healthy seeds of C. cajan cv ICP 26 were surface sterilized by agitating in screw cap bottle containing 70% ethanol for 1 minute, followed by 0.1% (w/v) (HgCl2) + Tween 20 (10 μl) for 7 min. Later these seeds were washed with sterile double distilled water for 4-5 times. Subsequently, the seeds were soaked in sterile distilled water for 14 h, drained aseptically, and rinsed thrice in sterile distilled water. Pre-soaked seeds were decoated and inoculated aseptically on MS/B5 medium for germination.
Culture media and culture conditions
Cotyledons from one-week-old in vitro grown seedlings were excised for initiating organogenesis and were inoculated on MS/B5 medium supplemented with different concentrations (0.5-5.0 mg/L) of BAP / KIN alone. The PH of the medium was adjusted to 5.8 either with 0.1 N NaoH or 0.1 N HCl before adding 0.8% (w/v) agar-agar and autoclaved at 121°C under 15 psi for 15-20 min. Cultures were maintained in a culture room at 25 ± 1°C under 16 h photoperiod at 50 μ mol m-2 s-1 provided by cool white fluorescent tubes (Philips, India) and 60-65% relative humidity.
In vitro rooting and plantlet establishment
The elongated shoots were transferred onto the MS medium supplemented with different concentrations of IBA (0.5-2.0 mg/L). The in vitro rooted plantlets were taken out from the culture vessels and washed with sterile distilled water to remove remains of agar. Later these were shifted to plastic cups containing vermiculite: soil (1:1). These cups were covered with polythene bag to maintain RH (80-90%) and kept in walk-in-chamber for acclimatization. After one month, these were transferred to pots containing garden soil and maintained in green house and finally transferred to the field.
Genetic fidelity of in vitro raised plants
Genetic fidelity of in vitro raised plants was carried out by RAPD [16] profiling. Genomic DNA was isolated from fresh and young leaves of four randomly selected in vitro regenerated plantlets of different replications as well as donar plant by the modified cetyl trimethyl ammonium bromide (CTAB) method [17]. For extracting genomic DNA, fresh leaves (1 gm) were grounded in liquid nitrogen, homogenized in 5 ml of extraction buffer (2% CTAB, 20 mM EDTA, 2% PVP) 1.4 M NaCl, 100 mM Tris-HCL pH 8.0 and 1% ß-mercaptoethanol) and incubated at 65°C for 1 hr. The supernatant was collected and treated with RNAse ‘A’ (100 μg/mL), and incubated at 37°C for 30 min and extracted twice with chloroform-isoamyl alcohol (24:1 v/v). Later the DNA was pelleted with ice cold isopropanol and washed 2-3 times with 70% ethanol. The pelleted DNA was air dried and stored at -20°C. DNA concentration was determined by using spectrophotometer.
PCR Amplification condition
RAPD (Operon Technologies Almeda, USA) primers (10) were used for screening of DNA to assess the genetic fidelity of in vitro regenerated plants. PCR [18] amplification was carried out in 20 μL reaction mixture containing 25 ng of DNA (2 μL), 2.0 μL of 10x thermostable PCR buffer with 15 mM MgCl2, 0.5 μL of 100 μM dNTP mixture, 2.0 μL of RAPD primers and 0.3 μL of Taq DNA polymerase (Bangalore Genei, India). PCR amplification was performed in a thermal cycler (Biorad, UK). The amplification programmes was as follows: an initial denaturation step at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing for 1 min at 37°C, extension for 2 min at 72°C, finally ending with one cycle for 5 min at 72°C. Amplified PCR products were resolved on 1.5% agarose gel electrophoresis with TAE buffer (1X) and ethidium bromide (EtBr). The gels were documented and analysed under gel documentation system (Bio Rad, UK). The amplified DNA bands were determined by using 1Kb molecular marker (Banglore Genei, India). 10 RAPD primers were selected to test genetic stability of randomly considered 4 regenerants after 3rd subculture cycle and the mother plant.
Data analysis
The data on induction and formation of multiple shoots were collected after 4 weeks of culture. The parameters such as percentage of response, mean number of shoots per explant, mean length of shoots, mean number of roots per explant and mean length of roots were recorded. The experiment was repeated at least thrice and data were statistically analyzed following the method [19].

Results

The results on in vitro direct regeneration from cotyledon explants of pigeon pea cv. ICP 26 from one week old axenic seedlings cultured on MS / B5 medium augmented with different concentrations (0.5-5.0 mg/L) of BAP / KIN alone are presented in Tables 1 and 2 respectively. Cotyledon explants cultured on MS / B5 medium without BAP/KIN did not induce the formation of multiple shoots. The cotyledons turned into green within a week on all the concentrations of PGRs used. In the present study, based on the type of medium and concentration of PGRs used, the number of adventitious shoots formation per explant was found to be varied in cotyledon explants.
Table 1: Effect of MS+BAP/KIN on in vitro direct regeneration from cotyledon explants of C. cajan.
Table 2: Effect of B5+BAP/KIN on in vitro direct regeneration from cotyledon explants of C. cajan.
Effect of MS medium
Cotyledon explants were cultured on MS medium fortified with various concentrations (0.5-5.0 mg/L) of BAP. Shoot bud formation was observed from cotyledon explants at the end of 2nd week of culture in all the concentrations of BAP. Maximum percentage (80%) of response and maximum number (38.1 ± 0.61 shoots/explant) of direct multiple shoot buds formation were observed on MS medium augmented with 2 mg/L BAP followed by 2.5 mg/L and 1.5 mg/L BAP (33.3 ± 0.03, 29.3 ± 0.16 shoots/explant) respectively (Figure 1a-e). On further increase in the concentration of BAP, a decrease in the number of multiple shoots was observed (Table 1).
Figure 1: (a-h): In vitro direct organogenesis via cotyledon culture in C. cajan cv ICP-26.
MS medium augmented with different concentrations of KIN showed less response in inducing multiple shoots from cotyledon explants compared to BAP. Shoot bud formation was observed from cotyledon explants at the end of 2nd week of culture in all the concentrations (0.5-5.0 mg/L) of KIN tested. Maximum percentage of response (78%) with maximum number of multiple shoots (33.1 ± 0.07) per explant was recorded at 2.5 mg/L followed by 2.0 mg/L KIN (30.6 ± 0.52) (Figure 1). On further increase in concentration of KIN, a decrease in the number of multiple shoots was observed (Table 1).
Effect of B5 medium
Cotyledon explants were cultured on B5 medium fortified with various concentrations of BAP (Table 2). Shoot bud formation was observed after 3-4 weeks of culture. Highest percentage of response (71%) with maximum number of multiple shoots (3.33 ± 0.17) per explant was observed at 2.0 mg/L BAP followed by 2.5 mg/L BAP (3.10 ± 0.52). On further increase in concentration of BAP, a decrease in number of multiple shoots from cotyledon explants was observed with an increase in the shoot length (Figure 1).
KIN did not induce multiple shoot buds in all the concentrations tested. Initiation of callus formation was observed by the end of 3rd week. Maximum percentage (82%) of response was observed at 5.0 mg/L KIN (Table 2). The percentage of response increased with increase in the concentration of KIN.
Plantlet establishment
The elongated micro-shoots (3-4.0 cm) were cultured on MS medium fortified with different concentrations (0.5-2.0 mg/L) of IBA (Table 3). The rhizogenesis was initiated within 2 weeks of incubation. Maximum percentage of root induction (80%) was observed at 1.0 mg/L IBA with an average number of 7 ± 0.36 roots/shoot (Figure 1f). The in vitro rooted plantlets were washed with sterile distilled water and transferred to plastic pots containing vermiculite: soil (1:1) and acclimatized in walk-in-chamber for 4 weeks (Figure 1g). Later they were shifted to earthenware pots containing garden soil and maintained in the green house. After 3 weeks they were shifted in to field. The percentage of survival of the regenerated plants was found to be 75% and the plants were similar with the parental plant by showing normal flowering and fruiting (Figure 1h).
Table 3: Effect of IBA on in vitro rooting of micro shoots in C. cajan cv ICP 26.
Genetic fidelity assessment
In the present study, the genetic fidelity of the regenerated plants was screened by RAPD markers that could detect the DNA sequence modifications at the primer annealing site of the genome. 20 primers (OPA 01-10 and OPD 01-10 series) were tested to analyze 4 regenerated plants as well as the donar plant. Among 20 primers tested, 7 primers (Table 4) responded for genomic DNA amplification and produced 15 bands. All of them were highly reproducible with molecular weight between 150-900 bp. All the responded primers revealed monomorphic banding pattern similar to the mother plant (Figure 2). OPA-09 and OPD-10 produced the maximum number of monomorphic amplicons (6), whereas OPA-03 produced the lowest number (1) of amplicon. Similar type of amplified amplicons was detected among all the micropropagated plantlets with reference to donar plant, which confirms the genetic stability of these in vitro derived plantlets.
Figure 2: Genetic fidelity analysis of in vitro developed plantlets through cotyledon culture of C. cajan using RAPD primer OPD-10. M- Marker; Dp- Donar plant; Lanes 1-4 (Randomly selected regenerated plantlets developed through cotyledon culture).
Table 4: Selected primers for genetic fidelity analysis in in vitro regenerated plants of C. cajan cv. ICP 26.

Discussion

In the present investigation direct regeneration has been successfully achieved on both MS and B5 media supplemented with various concentrations of BAP/KIN (Tables 1 and 2). Maximum percentage of response (80%) with maximum number of direct shoot buds were developed from the cotyledon explants on MS medium fortified with 2.0 mg/L BAP followed by 2.5 mg/L KIN. Whereas, less percentage of response was observed on B5 medium supplemented with all the concentrations of BAP/KIN tested.
As the concentration of BAP is increased, a decrease in the number of multiple shoots was observed. In the present study, of both the MS and B5 media used, MS medium was found to be more effective than B5 medium for direct regeneration from cotyledon explants of C. cajan.
Explants cultured on MS medium supplemented with varying concentrations of KIN evoked least response compared to BAP. BAP is more effective than KIN to trigger the synthesis of endogenous cytokinin in the present study [20].
Similar to our findings, regeneration of shoots from cotyledon explants on MS+ 2mg/L KIN was observed in C. cajan cultivar cv Hyderabad by Geetha et al. [6]. George and Eapen [7] reported the development of shoot buds from the distal end of the cotyledons on MS medium supplemented with 1 mg/L BAP + 0.1 mg/L IAA in pigeon pea. Similarly, Kumar et al. [13] have reported multiple shoots from whole cotyledons and nodal halves of cotyledons on MS medium supplemented with 0.5 mg/L 2,4-D + 2 mg/L BAP in C. cajan. Surekha and Arundhati [21] reported multiple shoots formation from cotyledon explants cultured on MS medium supplemented with 2.0 mg/L BAP + 0.1 mg/L KIN of three pigeon pea varieties ICPL 87, ICPL 85063, LRG 30. Similar results were also reported by Mehta and Mohan Ram [10] from cotyledons cultured on B5 medium supplemented with 105 M BAP in C. cajan. In the present investigations, it was interesting to note that callus was induced from the cotyledons cultured on B5 medium at all the concentrations (0.5- 5.0 mg/L) of KIN tested (Figure 1b). From the reports mentioned above it can be concluded that BAP is more effective for inducing multiple shoots in pigeon pea cv ICP 26.
Maximum percentage of rooting (80%) was observed at 1.0 mg/L IBA. Our results were similar to that of Mohan and Krishnamurthy [8]. Results have stated that 1.0 mg/l IBA induced maximum percentage of rooting in C. cajan, while, Eapen et al. [22] reported that 1 mg/l NAA induced maximum percentage (90%) of rooting in pigeon pea. In the present investigation, the protocol developed is useful for in planta transformation in C. cajan similar to the protocol developed by Yasmeen et al. [23] in Tomato and Keshamma et al. [24] in Cotton respectively by using Agrobacterium tumefaecians mediated genetic transformation.
Based on RAPD analysis the banding profiles from regenerated plants were found to be monomorphic and similar to those of the mother plant. This confirmed the true-to type nature of the in vitro raised plantlets of C. cajan cv ICP-26.

Conclusion

From the above study, it can be concluded that the regeneration of plantlets was achieved through direct organogenesis from cotyledon explants in C. cajan cv ICP-26 on MS medium supplemented BAP. Thus, the present reproducible regeneration protocol can be used for Agrobacterium tumefaecians mediated genetic transformation in C. cajan to transfer gene of interest.

Acknowledgements

Mrs. Mahitha Banala is grateful to ICRISAT, Hyderabad for providing germplasm of Pigeon pea cv ICP-26 and University Grants Commission, New Delhi for financial assistance as UGC-BSR Research Fellow (UGC Sanction Lr. No. F.4-1/2006(BSR) /7-211/2009(BSR); dt. 26-02-2013).

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