Development of an efficient regeneration system
Expedite seed germination process
To enhance germination rates and expedite germination process, various methods were employed.When seeds were directly cultured on MS basal agar medium in petri plate without any PGRs, germination ranged from 60-70% and took 5-7 days. However, placing seeds on sterile, moistened cotton (MS liquid) in jam bottles, germination reached 90-100% within 4-5 days period. Even more rapid germination (95-100% within 3-4 days) occurred when seeds were placed on sterile, moist filter paper soaked in MS liquid. Similar high germination results have been observed in other studies with various crop species using sterile, moistened filter paper or cotton in Petri plates or test tubes
(Geetha et al., 1998). Fastest germination (100%) was seen when seed was incubated in dark for 2-3 days on moistened filter paper in MS liquid with 2.5 mg/l BAP (Fig 1a).
Use of embryonic axes as explants
Axillary buds present at junction of cotyledon and embryonic axes contain meristem cells necessary for regeneration and are primary targets for gene delivery. After seed germination, embryonic axes were excised and decapitated by removing root and shoot meristem. Decapitated embryonic axes were placed on a preculturing medium (Fig 1b).
Optimized transformation process
Acetosyringone released in response to injuries in dicot plants, acts as a signaling molecule triggering transfer of T-DNA from
Agrobacterium to plant. Although acetosyringone and AB media have been commonly used in
in planta transformation technique, their use in
in vitro transformation of pigeonpea was not extensively documented
(Karmakar et al., 2019). Refinement of agro-infection parameters, such as bacterial optical density (OD), acetosyringone concentration, inoculation time and co-cultivation duration, would prove advantageous in enhancing
in vitro transformation efficiency. Eleven different treatment combinations were evaluated, each varying in bacterial OD600 (0.2, 0.5, 1.0), acetosyringone (100, 200 µM/ml), inoculation (15, 25 min) and co-cultivation (48, 72, 96 hrs) (Table 1). Optimal treatment combination for improved transformation efficiency was determined based on average number of explants recovered during kanamycin selection and average number of plants confirmed positive in
nptII PCR screening. Each treatment was replicated three times, with 50 explants per treatment. Optimized transformation parameters were found to be as follows: bacterial density in AB media (OD600) of 0.2, acetosyringone concentration at 100 µM, vacuum infiltration for 15 minutes and a 72-hour co-cultivation period, resulting in the highest (58-60%) transformation efficiency (Fig 5a).
Plant regeneration and acclimatization
Explants were rinsed, dried (Fig 1c) and moved to a selection medium that contained hormones and antibiotics. Kanamycin, an antibiotic, was added to facilitate selection and regeneration of transgenic shoots, while carbenicillin and cefotaxime were used to control growth of
Agrobacterium in plant culture. To minimize any adverse effects, a concentration of 250 mg/l for both carbenicillin and cefotaxime was chosen based on previous research
(Yu et al., 2001). Using kanamycin at 100 mg/l has been proven to be effective for selecting transgenic pigeonpea
(Ganguly et al., 2018). Agar is preferred solidifying agent for selection medium due to its ability to release kanamycin quickly, which reduces chances of escapes compared to using gelrite
(Krishna et al., 2011).
The ideal ratio of hormones plays a significant role in affecting enzymatic reactions in plants, influencing their growth and morphology. Various PGRs were chosen based on existing literature to develop a standardized protocol for regenerating and transforming two pigeonpea cultivars. Among cytokinins, BAP was preferred over kinetin (KIN) and thidiazuron (TDZ) for its ability to promote shoot bud regeneration. Past research has highlighted significance of BAP in facilitating both formation of callus and promotion of shoot growth, reducing regeneration time
(Thu et al., 2003). Explants were cultured on MS media (preculture and SMI) with different concentrations of BAP (2, 2.5, 5, 10 mg/l). In axillary bud regions, callus is induced when BAP concentrations were high (5.0 and 10 mg/l), but its growth was hindered. Few primordia-forming calli were detected at concentrations ranging from 2.0-2.5 mg/l. Small shoots, measuring 0.5-0.7 centimeters in length, with callus/explant, were moved to SMI that had varying levels of BAP (2.0, 2.5, 5.0 and 10 mg/l) to promote growth. It was found that BAP at 2-2.5 mg/l, was most effective in inducing shoot growth in about 3-4 weeks (Table 2). Results are consistent with earlier reports that found that cotyledonary node had high shoot bud regeneration at a BAP concentration of 2.0 mg/L
(Geetha et al., 1998; Thu et al., 2003). In most of calli, 70-80% of multiple shoots were produced, with each explant regenerating between 2 to 4 shoots (Fig 2). Excessive cytokinin (5.0 and 10 mg/l) results in intermediate callus formation, affecting regeneration. Addition of auxin along with cytokinin to enable both multiplication and elongation of shoots. Moreover, inclusion of GA3, along with other growth regulators, significantly enhanced occurrence of generating multiple shoots and subsequent generations of axillary nodes. Similar observations were also made in earlier studies
(Geetha et al., 1998; Sarkar et al., 2019). Here, we have prepared SMII with different concentrations of IAA (0.2-0.5 mg/l), GA (0.5-1 mg/l) and Zeatin (0.5-1 mg/l). Shoots-bearing explants were subcultured on SMII for shoot elongation. Most effective media for shoot regeneration were those supplemented with BAP 2.5 in preculture, BAP 2.5 in SMI, IAA 0.5, GA 0.5 and Zeatin 1 in SMII (Fig 1d and e). A maximum shooting frequency of 77-82% was achieved (Fig 5c).
For rooting, elongated shoots (2-4 cm) were removed and cultured on rooting media with various basal media, auxin and sucrose combinations. Sucrose concentrations of 1.5% and 2% were found to be most effective in promoting rooting, while external auxins further enhanced root development. Rooting media composition half MS + 20 g/l sucrose + 0.5 mg/l IBA was used because of its potential to generate 100% roots in pigeonpea variety JKPL
(Krishna et al., 2011). Whereas, hormone-free B5 medium was selected based on its rooting efficiency (>25%) for pigeonpea genotype ICPL 87
(Thu et al., 2003). According to our study, using half MS media with 15 g/l sucrose and 0.5 mg/l IAA resulted in better root growth for both genotypes and took around 3 weeks for roots to differentiate on elongated shoots (Fig 1f and g). A maximum rooting frequency of about 100% was achieved (Fig 5d). This finding is consistent with earlier studies on direct rooting
(Thu et al., 2003; Krishna et al., 2011).
Molecular analysis
PCR amplification using primers for
nptII and RT-PCR for
GUS was conducted (Fig 3 and Table 3). Out of 858 putative transformants of Pusa992 and 845 plants of Asha regenerated in kanamycin selection, 730 of Pusa 992 and 715 of Asha were positive through RT-PCR. Transformation efficiency was determined by dividing total RT-PCR positive transgenic lines by the number of explants used for transformation (Table 3). This resulted in a transformation efficiency of 57% for Pusa 992 variety and 65% for Asha. Similar transformation efficiencies using PCR analysis have been documented when embryonic axes were used as explants for transformation
(Thu et al., 2003).
GUS analysis
GUS assay was conducted to validate efficacy of transformation protocol. Initially,
in planta transformation was employed to ascertain optimal GUS treatment duration. After 3 days of co-cultivation, treatment durations of 4 hours, 6 hours and 12 hours were tested. Results demonstrated a progressive increase in GUS expression over time for both the varieties. Peak expression was observed after a 12-hour treatment (Fig 5b). Transformed tissues with GUS gene expression exhibited a prominent blue coloration, primarily concentrated in axillary bud region of
in planta transformed seeds due to activity of β-glucuronidase (Fig 4a i). In contrast, no GUS activity was detected in untransformed samples (Fig 4a ii). For tissue culture-based transformed explants, analysis was performed following a 3-day co-cultivation and subsequent 2 weeks of growth under selection conditions. Transformed explants displayed a positive reaction in assay (Fig 4b iii and iv). Untransformed exhibited a negative reaction, indicating absence of endogenous GUS-like expression (Fig 4b v). Findings indicated consistent integration and expression of GUS, implying stable integration of T-DNA within genome of primary transformants.
