Multiplication of Eucalyptus citriodora (L) Hook Through Shoot Bud Induction on the Internodal Portions of Mature Tree Explants

Nodal explants of Eucalyptus citriodora responded better for high frequency bud break and fast growth in liquid agitated cultures with 4.4 μM benzyladenine (BA) and 5.37 μM α-naphthalene acetic acid (NAA) rather than on semisolid medium. On transfer to MS medium with 1.11 μM BA and 5.37 μM NAA the sprouted axillary buds showed further elongation growth alongwith a slight callus and formation of globular structures on the internodal regions. The anatomy of these globular structures revealed that they are shoot buds. These buds elongated into shoots on MS medium with low levels of BA.     

Optimization of callus culture and shoot multiplication ofAsparagus densiflorus

The effects of different growth regulators on induction and growth of callus ofAsparagus densiflorus cv. Sprengeri were studied. Calluses grew more rapidly on Murashige and Skoog basal medium supplemented with 5.4 μM p-chlorophenoxyacetic acid (pCPA) and 4.4 μM 6-benzylaminopurine (BA) (medium 1) as compared to the same medium with 11.3 μM 2,4-dichlorophenoxyacetic acid (2,4-d) and 4.6 μM kinetin (medium 2). Calluses on medium 1 were soft and friable, whereas, compact, hard calluses originated on medium 2. Different concentrations and combinations of BA and/or kinetin were also used to study their effects on shoot regeneration. Kinetin was found to be less effective than BA in the initiation of shoots (1.8 shoots/callus). High numbers of shoots were produced in the presence of 0.4 μM BA alone (3.3 shoots/callus). The addition of ancymidol (5 μM) in MS with 0.4 μM BA enhanced multiplication of shoots (9.8 shoots/explant) and also produced well-developed crowns.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Artemisia abrotanum</Emphasis> L. by means of somatic organogenesis

The goal of this project was to regenerate Artemisia abrotanum L., Southern wormwood, by means of organogenesis from leaves. In vitro plant propagation may greatly support the molecular characterization of the medicinal qualities of A. abrotanum. Young, intact leaves were excised from mature plants and surface sterilized. Abundant callus growth, as well as shoot formation, was produced on an MS medium supplemented with 4.44 μM BA and 0.54 μM or 0.81 μM NAA. Shoots, with some residual callus, rooted equally well in MS media with 0.49 μM IBA, 0.54 μM NAA, or without hormones. Rooted plants were best acclimated in potting soil.     

Purple-leaved Ficus lyrata plants produced by overexpressing a grapevine VvMybA1 gene

Key message

This study established an efficient method of regenerating plants of Ficus lyrata and producing purple-leaved F. lyrata plants through genetic transformation using a VvMybA1 gene of grapevine.

Abstract

Ficus lyrata, a species with unique violin- or guitar-shaped leaves, was regenerated from leaf-derived calli cultured on Murashige and Skoog (MS) basal medium supplemented with 4.5 μM N-phenyl-N’-1, 2, 3-thiadiazol-5-yl urea (TDZ) and 0.5 μM α-naphthalene acetic acid (NAA). Leaf discs were inoculated with Agrobacterium tumefaciens strain EHA 105 harboring a binary vector DEAT that contains the VvMybA1 gene and neomycin phosphotransferase (npt II) gene and subsequently cultured on the established regeneration medium supplemented with 100 mg l^?1 kanamycin. Results showed that 87.5 % of the leaf discs produced kanamycin-resistant callus, and 68.8 % of them produced adventitious shoots. Transgenic plants with three leaf colors including green, green-purple, and purple were produced. Regular and quantitative real-time PCR analyses confirmed the integration of transgenes into the host genome. Semi-quantitative RT-PCR analysis indicated that the VvMybA1 gene was responsible for the purple-colored phenotype. Purple-leaved plants with strong color stability grew vigorously in a greenhouse. This study illustrated the feasibility of using a genetically engineered VvMybA1 gene for drastic modification of leaf color of an important woody ornamental plant.     

Interaction of Phytohormones in Regulating the Axillary Bud Growth in Pea

In the present study the interactions of GR24, a synthetic analog of newly discovered plant hormones strigolactones (SLs), with cytokinin (CK), benzyladenine (BA), auxin naphthaleneacetic acid (NAA), gibberellic acid (GA₃) and abscisic acid (ABA) in the regulation of axillary bud growth in pea (Pisum sativum L.) were investigated. The hormones were applied directly to buds at node 1 and 2 and the dose-response experiments were performed on 8–10-day-old SL-deficient rms1 and rms5–1 mutants, branching SL-sensitive rms2–1 mutants and wild-type plants. In the mutant plants the treatment with 5 μM GR24 completely inhibited bud growth while BA up to 100 μM stimulated it. The combined application of GR24 and BA showed that GR24 efficiency to reduce bud outgrowth constantly declines as CK-stimulated bud growth increased, with the inhibiting effect of GR24 abolished at 100 μM BA applied. GA₃ accelerated bud outgrowth, but did not interfere with GR24 inhibitory action. NAA reduced bud growth in intact SL-sensitive rms2–1 mutant and also in SL-insensitive rms3–2 and rms4–1 mutants. The NAA effect was observed already at 0.5 μM, however, even at a response saturating concentration of 500 μM its inhibiting effect was inferior to that of 5 μM GR24. At combined treatment the effectiveness of GR24 in suppressing bud growth decreased with a decrease in NAA-inhibited bud growth, suggesting that auxin might act as a suppressor of SL action. ABA strongly inhibited the bud outgrowth and, like CK and auxin, reduced the inhibitory effectiveness of GR24. The revealed ability of CK, ABA and auxin to suppress bud response to SLs is supposed to result from phytohormone signaling crosstalks.     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Efficient In vitro Plant Regeneration Protocol from Leaf Explant of Jatropha curcas L — A Promising Biofuel Plant

An efficient in vitro plant regeneration from leaf-disc culture of Jatropha curcas L has been established. Adventitious shoot buds along with callus were induced from leaves of 2-year-old J. curcas plants cultured on Murashige and Skoog’s (MS) medium supplemented with TDZ (2 μM) BAP (2 μM) and IBA (1 μM), wherein 63.3% leaf explants responded. The multiplication of shoots was achieved from the adventitious shoot buds after transferring them to shoot induction medium. The highest number of shoots (9.7/explant) was achieved after 6 weeks of culture on MS medium containing 3 μM of BAR The welldeveloped shoots were rooted on MS medium supplemented with IBA (1.5 μM) with the rooting frequency of 53.3%. Addition of phloroglucinol (200 μM) to the medium enhanced the frequency of rooting to 76.7%. Regenerated plantlets were successfully transferred to field after initial acclimatization.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

High Frequency Clonal Propagation of Cymbopogon martinii var motia (palmarosa) Through Rhizome Culture and True to Type Assessment using ISSR Marker

A rapid and high frequency plant propagation system has been established in Cymbopogon martinii var motia using rhizome culture. Different concentrations of auxins, such as 5.36 μM Napthalene acetic acid (NAA) and 5.71μM Indole 3acetic acid (IAA) satisfactorily induced shoot buds when applied individually or in combination with 4.40μM N⁶-Benzyladenine (BA) on MS medium. Multiple shoot proliferation was noted when induced microshoots were cultured on MS medium supplemented with BA either alone or with NAA. Shoot bud induction and multiple shoot formation were enhanced significantly with the application of growth additives like coconut water (CW) and biotin. Out of the two auxins (IAA and IBA) tested, rooting percentage and number of roots/shoot was significantly higher on 1/2 MS medium supplemented with 4.90 μM Indole 3- butyric acid (IBA). Twelve reproducible ISSR primers efficiently screened 16 randomly chosen regenerants of C. martinii with similar monomorphic banding profiles exhibiting genetic stability of the regenerants.     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

Effects of culture conditions on multiple shoot induction from inflorescence and RAPD analysis of cloned plants in Limonium sinensis (Girard) Kuntze, var. Golden Diamond

The optimum physical and chemical microenvironment for micropropagation of Limonium sinensis (Girard) Kuntze, var. Golden Diamond was established from immature inflorescence segments as explant. The highest frequency (62 %) of axillary shoot induction was obtained on MS medium (Murashige and Skoog Physiol Plant 15:473–497, 1962) supplemented with 8.88 μM BA, 1.34 μM of NAA and two growth additives cysteine (142.33 μM), and glutamine (684.22 μM). In the subsequent culture maximum average number of shoots (11.13?±?0.34) were obtained from micro-shoot explant on MS medium supplemented with the same additives and 2.22 μM BA. During subcultures the problem of vitrification was mitigated through increasing agar concentration from 0.8 % to 1.0 % and providing better ventilation. The in vitro developed shoots were rooted on MS medium supplemented with 2.46 μM IBA and 0.88 μM BA. Rooted plants were acclimatized successfully in the greenhouse with 80 % survival rate. RAPD analysis using 15 random decamer primers generated monomorphic banding pattern in micropropagated plants and similar to those of mother plant revealing the genetic integrity of regenerants.     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

In vitro differentiation in callus cultures of moth bean,Vigna aconitifolia (JACQ) marechal

Callus cultures of two cultivars of Vigna aconitifolia (IPCMO-926, RDM-120) were raised and their growth and differentiation studied. In IPCMO-926 callus cultures, numerous shoot buds differentiated on MS medium with BA (0.4–22.2 μM) alone or in combination with IAA (5.7 μM). In RDM-120 best differentiation of shoot buds was observed on a medium with K (23.2 μM) and IAA (5.7 μM). Kinetin alone, however, induced rhizogenesis in callus cultures. In suspension cultures of IPCMO-926 embryoids differentiated on MS medium with K (0.5 μM) and 2,4-D (0.4 and 0.9 μM).     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.