High Frequency Plant Regeneration Research Articles

Development of regenerated plants of interspecific wheat hybrids using immature embryos as explants

Nowadays particular attention in winter wheat breeding is paid to the introduction of biotechnological methods. The culture of immature embryos in vitro is an alternative way to produce hybrids that often do not develop fully in vivo. The purpose of the current study was to estimate the ability of immature embryos of interspecific winter wheat hybrids for callus induction, morphogenesis, and plant regeneration on artificial nutrient media, as well as to select optimal conditions for obtaining full-fledged plants with all the botanical characteristics of the species. The research material included 5 hybrid combinations of interspecific crosses between durum (Triticum durum DESF.) and common (Triticum aestivum L.) winter wheat. The material was collected during one field season (2022). Immature embryos were used as explants. Culture media were according to Murashige-Skoog (MS), and they differed in the content of hormones and organic substances. There has been found that the hybrid combinations were characterized by a variety of morphogenic responses. The manifestation of callusogenesis was not high enough and averaged 22–32 % depending on the nutrient medium. Rhizogenesis averaged from 1–6.7 %, and ended with the formation of roots. The process of formation of embryoid- and hemmorizogenesis was best demonstrated by the hybrid combination No.1, and on two media. All processes of morphogenesis excepting ‘rhizogenesis’ varied greatly among hybrid combinations. The largest number of regenerated plants was obtained from hybrid combination No.1 (61 pcs., 54.5 %). After vernalization and acclimatization, 38 % of the plants survived from the number of planted embryos. The largest number of heads (79 pcs.) was formed according to hybrid combination No.1, on average 1.46 heads per plant. However, not all heads were fertile. The results of the correlation analysis made it possible to identify indicators that closely correlated with the amount of plant regeneration, namely, the high yield of embryoid- and hemmorizogenic structures ensured a high frequency of plant regeneration (r = 0.769).

Optimization of high frequency plant regeneration protocol via embryogenic callus formation from diverse indica rice genotypes induced by combinations of 2,4-D and BA

A high frequency plant regeneration protocol via induction of embryogenic callus followed by somatic embryogenesis was standardized for a group of indica rice varieties consisting of land races and improved varieties, which are generally considered recalcitrant. Scutellum derived embryogenic calluses were obtained from the mature seeds inoculated into MS medium supplemented with different concentrations and combinations of 2,4-D and BA added with casein hydrolysate (500 mg/l) and proline (1 g/l). The varieties significantly differed in their response to embryogenic callusing and regeneration. Maximum embryogenic callus formation was induced in all the varieties by altering the combinations and concentrations of 2,4-D(1.0-2.0 mg/l) and BA (0.01, 0.1 and 0.5 mg/l). The land races viz., Karnel, SavuluSamba and Doddabyranellu showed higher embryogenic callus formation in all the combinations and concentrations of 2,4-D and BA. But the improved varieties showed embryogenic callus induction only in particular concentration and combinations of growth regulators. However, an increasing trend of plant regeneration was observed in regeneration media consisted of MS salts supplemented with 3.0 mg/ l BA and 1.0 mg/l NAA. We showed that maximum plant regeneration through generation of embryogenic calluses induction in indica rice varieties can be induced by altering the concentrations and combinations of 2,4-D and BA.

A simple protocol for high frequency plant regeneration and enhancing Shikonin production from callus cultures in Arnebia hispidissima

A rapid and efficient protocol for callus induction, enhancing Shikonin pigment production and plant regeneration from various explants such as leaf, shoot tip, internodal and nodal explants is presented. Highest callus induction efficiency of 86.7% per explant was obtained with nodal explants on MS medium blended with 0.5 mgl−1 KIN, 1.0 mgl−1 2, 4-D and 100 mgl−1CH medium. Callus from nodal explants grew faster and showed improved potentiality for shoot regeneration (80%) on MS medium supplemented with 0.5 mgl−1 KIN, 0.25 mgl−1 BAP, 0.1 mgl−1 IAA and 100 mgl−1 CH. MS medium added with 2.0 mgl−1 IBA efficiently induced roots in proliferating shoots further those were transferred to soil. Greatest response obtained for callus induction for Shikonin production was 80.0% from nodal segments, 46.7% from shoot tip explants followed by an intermediate response of 66.7% from internodal and leaf segment cultured on media combination MS + mgl−1 BAP 0.25 + 1.0 mgl−1 IAA + 100 mgl−1 CH. Shikonin pigment production was stimulated upon transfer of callus tissues to shoot induction medium containing IAA, BAP and KIN instead of 2, 4-D under 12 h dark period. MS medium supplemented with 0.25 mgl−1 BAP, 1.0 mgl−1 IAA and 100 mgl−1 CH was found to be ideal for high Shikonin induction in callus cultures of all the explants employed. UV–visible spectrophotometry quantified Shikonin content of 0.50 mgg−1 in fresh callus after 50 days of culture. Thus, a reproducible protocol for establishment of fast growing callus with high frequency plant regeneration from nodal segments of Arnebia hispidissima which could serve as a reliable source for enhanced production of Shikonin for pharmaceuticals, medicinal and food industry has been presented.

Callus-Mediated High-Frequency Plant Regeneration, Phytochemical Profiling, Antioxidant Activity and Genetic Stability in Ruta chalepensis L.

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 μM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 μM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 μM (BA) and 1.0 μM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 μM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

High throughput anther culture response in an upland rice cross ‘Khandagiri x Dular’

Aim: The experiment was conducted to maximize anther culture response in an upland indica rice cross amenable for drought tolerance breeding. Methodology: The cold pre-treated anthers of a cross Khandagiri (drought sensitive) x Dular (drought tolerant) were cultured on N6, SK3, MS and CIM media at varying concentration of hormones (2,4-D, Kn and NAA) to assess callusing response. Embryogenic calli were placed on regeneration medium (RM) with varying concentration of Kn, BAP and NAA for plant regeneration. The plantlets were acclimatized in half-strength MS basal liquid medium for one week before transferring to pot mixture (peat moss: perlite 2:1, v/v) for plant establishment in glasshouse under partial shade. Finally, the plantlets were checked for doubled haploid status by cytological study of root tips. Results: Anther culture response was found to be media and genotype-specific. F1-progenies responded better than either of the parents. A modified MS callus induction medium (CIM) with 1.5mg/l 2,4-D + 0.5mg/l Kn resulted impressively higher callusing response (30.2%) with nodular calli than SK3>MS>N6. High frequency (12.8%) of albino-free green plant regeneration with well developed rooting was achieved in RM medium (a modified MS) containing 2mg/l BAP and 0.5mg/l NAA. A total 129 doubled haploid (DH) plants (each with 12 pairs of chromosomes) were recovered which maintained normal growth, set seeds and resulted in true breeding DH lines. Interpretation: The high throughput regeneration system is amenable for doubled haploid production from indica crosses. Besides, the present doubled haploid stock can serve as an ideal mapping population and as such targeted for marker aided selection for early development of drought tolerant rice varieties.

English

Sorghum is a tropical grass used for various purposes including food, animal feed, and biofuels source, but its in vitro propagation still found an obstacle due to tissue browning. This study aims to propagate and regenerate callus in the Indonesian sorghum hybrid, named Numbu cultivar from seed and meristematic leaf whorl explants. The combined effect of plant growth regulators (PGRs), namely 2,4-D and proline as well as NAA (naphthalene acetic acid) and BAP (6-benzyl amino purine) was investigated on embryogenic callus induction and regeneration, respectively. The results showed that callus induction was significantly faster in the seed explants compared to leaf whorl. The optimal medium used to obtain a large induction in the seed explants was MS salt containing 3 mg L-1 2,4-D and 5000 mg L-1 proline, which gave a 68% yield. Furthermore, the best medium used to obtain a high plant regeneration frequency was the MS medium containing 0.1 mg L-1 NAA and 3 mg L-1 BAP, which regenerated 78% of the callus into plantlet. The addition of 560 mg L-1 proline to the combination medium increased doubled the regeneration into the planlet. Based on the results, PGRs combinations are required to improve embryogenic callus induction and regeneration into planlet in sorghum. © 2022 Friends Science Publishers

Thidiazuron induces high-frequency plant regeneration from shoot tip explants of an ornamental aquatic plant, Anubias barteri var. nana

ISHS IX International Scientific and Practical Conference on Biotechnology as an Instrument for Plant Biodiversity Conservation (physiological, biochemical, embryological, genetic and legal aspects) Thidiazuron induces high-frequency plant regeneration from shoot tip explants of an ornamental aquatic plant, Anubias barteri var. nana

Synergetic effects of TDZ with various phytohormones on high-frequency plant regeneration from mature nodal explants of Capparis decidua and their ex vivo implications

Synergetic effects of TDZ with various phytohormones on high-frequency plant regeneration from mature nodal explants of Capparis decidua and their ex vivo implications

High Frequency Plant Regeneration from Leaf Explants of Plumbago indica L.

High Frequency Plant Regeneration from Leaf Explants of Plumbago indica L.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function as opposed to the allotetraploid cotton species. As a prerequisite for this, a reliable and efficient method of high frequency plant regeneration in G. arboreum must be established. In this study, callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB medium (MS salts and B5 vitamins) with 0.09 µM 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 2.32 µM KT (kinetin). During propagation, callus was effectively selected for subculture on different media based on cell morphology to induce embryogenic callus and somatic embryos. Embryogenic callus was induced on MS5 medium (MSB, 37.59 mM KNO3, 62.47 µM NH4NO3, 3% (w/v) glucose, 6.8 mM glutamine, 3.8 mM asparagine) from suspended cultures after several cycles of alternate solid MS3 medium—liquid MS4 medium culture, a key step in somatic embryogenesis. Solid MS3 medium was supplemented with 2.46 µM IBA (īndole-3-butyric acid), 0.93 µM KT, 6.8 mM Gln, 3.8 mM Asn; liquid MS4 medium was supplemented with 0.1 g/L NaCl, 0.1 g/L KCl and 0.1 g/L CuSO4. The solid–liquid alternate culture was effective for embryogenic callus induced in G. arboreum. MS5 medium with maltose (3% w/v) or glucose (1.5% w/v) + maltose (1.5% w/v) performed better than that added single glucose (3% w/v) for somatic embryo maturation and germination. The regenerated plants with well-developed roots were directly transferred to the soil or grafted onto germinated cotton plants. Plant regeneration via somatic embryogenesis in diploid cultivated species was established, but there is a need for improvement and optimization for gene functional analysis and gene editing in the diploid cotton species. Regenerated plants of G. arboreum, a cultivated diploid cotton, were obtained via somatic embryogenesis by adjusting the culture mode and medium compositions.

High-frequency plant regeneration and genetic homogeneity assessment of regenerants by molecular markers in turmeric (Curcuma longa L.)

High-frequency plant regeneration and genetic homogeneity assessment of regenerants by molecular markers in turmeric (Curcuma longa L.)

Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos

BackgroundTrachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg’s B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. ResultsAccording to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. ConclusionsIn this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.

Callus Induction and Regeneration from Anther Cultures of Indonesian Indica Black Rice Cultivar

The assembly of superior varieties and collection of rice germplasm involves the process of selecting and storing elders that have superior genotypic properties and phenotypes. The anther culture techniques on indica black rice cultivar have a high difficulty factor to get plants, because of the low regeneration ability at the plant formation phase from the anther callus. This study aimed to investigate the influence of the cold-pretreatment time on anther, the combination of plant growth regulators (PGR’s) concentrations, and putrescine concentrations in media for the increase callus induction and plant regeneration of indica black rice. The optimization of the cold pre-treatment time was important to obtain the high-frequency callus induction, which showed that anther at the 4°C for 8 days formed the high callus induction (20%). To accelerate the callus induction, the application of 20 µM putrescine in the MS medium could produce more friable embryogenic callus for 24 days with 27% of callus formation. Generally, the optimal medium for the high frequency of callus induction contained 2 mgL−1 NAA+0.5 mgL−1 Kinetin+20 µM putrescine. Especially indica black rice cultivars, the best media to get a high plant regeneration frequency were N6 media containing the combination of 2 mgL−1 IAA and 2,5 mgL−1 Kinetin. The total callus regenerated to plantlet about 12.5%. The study of the callus induction and in-vitro plant regeneration medium for indica black rice were still important to develop to get the best result for other cultivars.

High frequency plant regeneration from leaf culture of Neolamarckia cadamba.

Neolamarckia cadamba is a miracle tree species with considerable economic potential uses as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish a highly efficient and robust protocol of plant regeneration for N. cadamba. Greenish callus was induced from very young leaf explants of sterile in vitro plantlets cultured on Murashige and Skoog's (MS) medium supplemented with 3 mg l-1 thidiazuron (TDZ), 0.1 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg l-1 α-naphthaleneacetic acid (NAA). The callus could differentiate into nodular embryogenic structures or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. The micro-shoots developed roots in MS supplemented with 0.05 mg l-1 NAA and 0.05 mg l-1 indole-3-butyric acid (IBA), while the nodular embryogenic structures germinated directly and developed into plantlets on induction medium contained with 0.5 mg l-1 (or 1 mg l-1) 6-benzyladenine (6-BA) and 0.05 mg l-1 NAA. The rooted plantlets could be successfully acclimatized to a greenhouse with more than 92.0% survival. This regeneration protocol can be used in large scale cultivation needs and may be useful for future genetic modifications of N. cadamba.

Establishment of a high-frequency plant regeneration system from rhizome-derived embryogenic cell-suspension cultures of Curcuma longa L.

We developed an efficient plant regeneration technique for turmeric (Curcuma longa L.) via somatic embryogenesis derived from embryogenic cell suspensions cultured from petioles. We initiated in vitro callus cultures from the plantlets of turmeric cultured on Murashige and Skoog (MS) medium. Then leaf, petiole, stem, and root explants of in vitro-grown plantlets were cultured on Schenk and Hildebrandt (SH). The stem and root explants formed white nodular calluses at a frequency of 86.7% and 85.8%, respectively, when cultured on SH medium; however, we used the calluses derived from the root explants for initiating a cell-suspension culture. In this study, the actively growing cell suspension culture comprised small, highly cytoplasmic cell aggregates and large vacuolated cells. On being transferred to a basal SH medium (without growth regulators), a few of these calluses differentiated into somatic embryos, which indicated that the initial calluses were embryogenic cells. We stimulated additional shoot differentiation by adding 0.5 mg L−1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) and various concentrations of N6-benzyladenine (BA) to the embryogenic cell suspension. The highest frequency at which shoot primordia developed from embryogenic cell clusters was 70% (when cultured in the dark on the supplemented MS medium), with more than 83.3% of regenerated shoot primordia producing roots. We successfully transplanted rooted plantlets into a soil mixture of sterile vermiculite and potting soil (1:1) and grew them to maturity in a growth chamber, achieving a survival rate of > 95%. To our knowledge, this is the first report of complete plant regeneration of turmeric from embryogenic cell-suspension cultures. The embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and gene manipulation for improving turmeric quality.

In vitro plant regeneration of wild eggplant (Solanum sisymbriifolium) to produce large number of rootstocks for tomato grafting

The experiment was conducted to develop a suitable protocol for high frequency plant regeneration of wild eggplant (Solanum sisymbriifolium) in order to produce a large number of rootstocks for tomato grafting for the management of wilt disease. To obtain in vitro seedlings of S. sisymbriifolium, seeds were treated with various concentrations of GA3 (Gibberellic acid) prior to place them in germination media (½ strength MS) and 750 mg/L GA3 was found as a suitable concentration resulting the highest (76.67%) germination rate. Various factors namely combination of plant growth regulators, explant types and explant age were investigated for development of an efficient plant regeneration system of S. sisymbriifolium. Cotyledon and hypocotyl explants of S. sisymbriifolium were cultured on MS medium supplemented with various concentrations of BA (6-Benzylaminopurine), NAA (α-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxy acetic acid), to determine suitable medium for callus and shoot initiation. Fourteen days old cotyledon explants were found more responsive than that of hypocotyl, both in callus and shoot induction. The highest callus initiation (100%) and shoot regeneration (73.33%) were observed in MS media supplemented with 0.5 mg/L NAA + 1.0 mg/L BA and 0.2 mg/L NAA + 3.0 mg/L BA, respectively. MS medium supplemented with 0.1 mg/L NAA showed the highest frequency (86.67%) of rooting. The regenerated plantlets were acclimatized in pot soil and eventually used as rootstock for tomato (Solanum lycopersicum cv. BARI hybrid 4) grafting. The grafted plants showed no wilt disease in field condition until maturity.

High-frequency plant regeneration from embryogenic cell suspension cultures of Gynura procumbens

The efficient plant regeneration system from embryogenic cell suspension cultures of Gynura procumbens (Lour.) Merr. is described. Leaf, stem and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) in various concentrations (0, 0.1, 0.3, 1.0 and 3.0 mg l−1). Leaf, stem and petiole explants formed pale-yellow nodular callus and off-white calluses at a frequency of 100% when cultured on MS medium supplemented with more than 1 mg l−1 of 2,4-D after 4 weeks incubation. However, only 20% of pale-yellow nodular callus derived from petiole explants developed into white embryonic structures. Upon transfer to MS basal medium without growth regulators, these white embryonic structures differentiated into somatic embryos. Embryogenic cell suspension cultures were initiated from petiole-derived pale-yellow nodular calluses. More than 73.2% of regenerated plantlets via somatic embryogenesis produced roots on MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid and 1 mg l−1 indole-3-butyric acid (IBA), respectively. Rooted plantlets were successfully transplanted to soil mixture of sterile vermiculite and potting soil (1:1) and grown to maturity in a growth chamber, achieving a survival rate of > 95%. The plant regeneration system from embryogenic cell suspension cultures of G. procumbens established in this study could be applied as an alternative for mass proliferation as well as molecular breeding for quality improvement of G. procumbens.

High frequency plant regeneration from cotyledonary node explants of Cucumis sativus L. cultivar ‘Green Long’ via adventitious shoot organogenesis and assessment of genetic fidelity by RAPD-PCR technology

Influence of cytokinins, silver nitrate (AgNO3) and auxins on plant regeneration from cucumber was investigated. The cotyledonary node explants were cultured on MS medium augmented with various concentrations (0.5-2.5mg l-1) of 6-benzyl amino purine (BAP) and kinetin (KIN) for shoot bud induction. BAP at 1.5mg l-1 was found to be the best concentration for induction of high frequency of multiple shoots (98.4%). Interestingly, maximum percent of multiple shoot regeneration (100%) as well as number of shoot buds (54.6 shoots/culture) was recorded on MS medium containing the combination of 4.5mg l-1 AgNO3 and 1.5mg l-1 BAP. Multiple shoot bud regeneration frequency as well as the number of shoots was positively correlated with the concentrations of AgNO3. Addition of silver nitrate in the medium not only enhanced the rate of multiple shoot bud regeneration but also elongation of shoot buds was observed. The highest percent of rooting (96.2%) was noticed on a medium containing the combination of indole 3-butyric acid (IBA), 1.5mg l-1 and KIN 0.5mg l-1. Acclimatized plantlets were successfully established in the field where the survival rate observed was 72%. The RAPD profiles of in vitro regenerated plants were found to be highly monomorphic and identical banding pattern with mother plant. DNA fingerprinting results confirmed that the tissue culture plantlets were found to be true-to-type. The present study describes efficient protocol for high frequency plant regeneration via adventitious shoot organogenesis in cucumber.

In vitro callus induction and regeneration of multiple shoots through callus derived from leaf and epicotyl explants in pigeon pea (Cajanus cajan L.)

An efficient in vitro protocol was developed for callus induction, high frequency plant regeneration through callus cultures derived from cotyledonary leaf and epicotyl explants, rooting of shoots derived from callus and establishment onto the natural conditions in two cultivars of pigeon pea; ICPL 87119 and ICPL 8863. Cotyledonary leaf and epicotyl explants were tested for callus induction across 48 different combinations and concentrations of auxins and cytokinins in MS medium, wherein, higher doses of auxins (15 mg/1 NAA) in combination with lower doses of cytokinins (0.5 mg/l kinetin) induced regenerable callus from leaf explants while lower doses of auxins (0.2 mg/1 NAA) in combination with higher doses of cytokinins (8 mg/1 kinetin) induced regenerable callus from epicotyl explants in both the genotypes. Plantlet regeneration from leaf and epicotyl derived callus was optimized at 0.05 mg/l TDZ in both genotypes. Rooting was optimized on ½ MS + 0.5 mg/1 IBA media in both genotypes. Well-rooted plants were acclimatized and established successfully into natural conditions in potting mixture-containing soil: FYM in 1:1 ratio resulting in 48.01 per cent survivability. Regenerated plants were uniform morphologically with normal leaf shape and growth. This protocol finds its significance in rapid multiplication of transgenic plants.