Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson

Prolific shoot bud differentiation was induced in callus and suspension cultures of hypocotyl origin in Brassica campestris cv. Yellow Sarson on MS medium supplemented with K (13.9–23.2 M) or BA (13.3–22.1 M). Plantlets were obtained by rooting the in vitro differentiated shoots. Histological studies revealed a unique mode of meristemoid formation.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA Benzyladenine - IAA Indole-3-acetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthalene acetic 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.     

Genetic transformation ofEustoma grandiflorum byAgrobacterium tumefaciens

Callus cultures were initiated from micropropagated Artemisia absinthium plantlets on MS basal medium supplemented with different concentrations of BA, Kn, NAA, IAA and 2,4-d in combination or singly. Supplementing the medium with low doses of both BA in combination with NAA, and Kn in combination with NAA enhanced the growth rate of callus cultures. However, cultures grew slowly following the second subculture and the majority turned brown and died within the next month. Initiation of root and shoot primordia occured directly from leaf explants cultured on 1.81 M 2,4-d, while adventitious shoot formation from callus was observed occasionally when BA was added to the medium in combination with IAA. Furthermore, medium containing 2.22 M BA and 2.69 M NAA stimulated both callus growth and organogenesis on some callus cultures derived from leaves and stems of young stock material. The best results were obtained with leaf explants. Cytological analysis of root meristems revealed that all regenerants were diploid (2n=18), as expected.Abbreviations MS Murashige & Skoog's salts and vitamins (1962) - BA 6-benzyladenine - NAA alphanaphthaleneacetic acid - Kn Kinetin (6-furfurylaminopurine) - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - FW fresh weight - Bi biomass increase     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

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.     

Growth and differentiation of callus cultures of Pinus

Segments of hypocotyl and cotyledons of aseptically-grown seedlings of Pinus strobus L. (white pine) and P. echinata Mill (shortleaf pine) were used as explants for establishing tissue cultures. Growth and differentiation of callus were studied on a modified Murashige and Skoog's medium containing nutrients and plant growth regulators. Meristems below the surface of callus tissue of P. strobus could be induced on media supplemented with -naphthaleneacetic acid alone or in combination with certain other plant growth regulators. Occasionally, differentiation of shoot buds also occurred on callus cultures. These shoot buds could be grown in vitro but roots did not develop.Abbreviations ABA abscisic acid - BA 6-Benzyl-aminopurine - 2-ip N⁶-(²-isopentanyl)-adenine - GD Gresshoff and Doy's medium - GE Gamborg and Eveleigh's medium - MS Modified Murashige and Skoog's medium - NAA -naphthaleneacetic acid - SC Sommer and Caldas' medium - TIBA 2,3,5-Triiodobenzoic acid     

Production of triploid plants from endosperm cultures of Phlox drummondii

Triploid plants of ornamental Phlox drummondii Hook. were raised from cultures of endosperm excised from immature fruits having zygotic embryo at early dicotyledonous stage. Endosperm tissue was firstly cultured with the embryo on the Murashige and Skoog’s (MS) medium supplemented with 5 μM 6-benzylaminopurine (BAP) + 10 μM α-napthaleneacetic acid (NAA) for 7 d and recultured after the embryo was removed. A friable callus appeared two weeks after removal of the embryo and it became compact callus mass in another three weeks. Upon transfer of this 5-week-old callus to the MS medium with 10 μM BAP + 2.5 μM indole-3-acetic acid (IAA), maximum percentage of green nodular shoot buds appeared from which regenerated dwarf shoots. Elongation of the dwarf shoots, however, required transfer of the individual dwarf shoots excised from the callus on the fresh medium and best results achieved on medium with low concentration of IAA (0.5 μM) in presence of 10 μM BAP. The shoots were then rooted in vitro and plants subsequently established in pots containing soil. Over 70 % of plants were triploid with a chromosome number of 2n=3x=21. Size of stem, leaves, flowers, pollen, and stomata of these triploid plants were higher and the plants were more vigorous as compared to naturally occurring diploid plants. In particular, flowers showed bright colour with enlarged central eye adding to their ornamental value.     

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.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Induced Caulogenesis in Long-term Callus Cultures of Rosrnarinus officinalis L

The callus formed in Rosmarinus officinalis L in association with shoot tip proliferation was isolated and subjected to different treatments for good growth. Two basal media, namely, Murashige and Skoog (MS) and Schenk and Hildebrdndt (SH) and their modifications supplemented with 0.25 mg I^-1 6-benzylaminopurine (BAP), 0.5 mg I^-1 indole-3-acetic acid (IAA) and 1.0 mg I^-1 2,4-dichlorophenoxyacetic acid (2,4-D) were used. Callus in MS medium, was compact and remained fresh and green upto 30 days but grew slowly. Whereas, in SH medium callus growth was rapid but it turned brown within 15 days.The browning of callus could be checked with the addition of 1500 mg I^-1 NH,NO, to the medium, in which callus grew 15 fold in fresh weight during 21 days and remained fresh upto 45 days of incubation.The shoot buds differentiated in this somatic callus with the addition of 0.5 mg I^-1 each of BAP, 2-isopentenyl adenine (2ip), IAA and 10 mg I^-1 gibberellic acid (GA₃), within 15 days of incubation provided the callus remained floating on the liquid medium. Histological investigations revealed both peripheral and occasionally internal differentiation of shoot buds. Differentiated shoot buds were proliferated, rooted and transplanted in the soil.     

In vitro clonal multiplication of mature trees of Dalbergia sissoo Roxb.

Multiple shoots were obtained from nodal explants of 30-year-old trees of Dalbergia sissoo Roxb. on a defined medium, MS (Murashige & Skoog medium) supplemented with auxin-cytokinin combinations. IAA (Indole accetic acid) alone promoted 15% rooted shoot buds. A combination of IAA+Kn (Kinetin) gave 100% rooted shoot buds. A combination of NAA (napthaleneacetic acid) + Kn and NAA+BAP (6-benzylaminopurine) also gave high percentages of rooted shoot buds. Ascorbic acid in the medium prevented the death of callus and plantlets, which followed darkening of the medium.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Plant regeneration from mesocotyl callus of Hordeum vulgare L.

Callus tissue was induced on barley mesocotyl explants of germinated seven-day-old seedlings on MS medium supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically different tissue cultures were maintained in vitro for a long time: a callus tissue without organogenesis and a culture with high rhizogenic capacity. Shoots and plantlets were generated when the auxin-media induced callus was transferred to medium supplemented with 3 M TIBA. In 62% of cultures, during the first five subcultures, four to twentyeight plants per single mesocotyl were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one mesocotyl was forty. Plantlets rooted well on MS with 5.7 M IAA and survived transplantation to soil in high percentages.Abbreviations IAA indole-3yl-acetic acid - BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog (1962) medium     

Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia

Micropropagated shoots of Maytenus ilicifolia Mart. were obtained from axillary buds cultured in Murashige & Skoog medium supplemented with 13.3 M 6-benzyladenine (BA). Addition of 1.1 M 1-indole-3-acetic acid (IAA) to the medium increased shoot elongation. The number of shoots formed was influenced by BA concentration, degree of juvenility of the explant, and by bud explant position on the stem. Cultures of buds taken from stem parts located close to the shoot tip yielded more callus than shoots, whereas axillary buds at distant positions from the apical bud yielded more shoots.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic-acid     

Efficient micropropagation protocol of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. possessing strong antimalarial activity

Micropropagation has been achieved in a promising larvicidal asteraceous taxon Spilanthes acmella L. using seedling leaf explants. The explants were reared on a variety of growth regulators, namely 2,4-dichlorophenoxyacetic acid, 1-naphthalene acetic acid, Indole-3-butyric acid, N⁶-benzyladenine, and kinetin either alone or in combination on Murashige and Skoog’s (MS) medium. The best green and compact callus was obtained on 1 μM NAA and 10 μM benzyladenine (BA) in 15 d. The callus on subculture to the same but fresh medium after every 30 d differentiated an average of 12.90 ± 0.32 shoot buds in 50% cultures. Elongation in shoot buds occurred only if they were transferred to NAA lacking MS+BA medium. An average number of 4.22 ± 0.83 shoots and 15 ± 0.84 shoot buds per explant were obtained in 70.3% cultures on MS + 10 μM BA in 30 d. One hundred percent excised shoots rooted in MS(1/2) + 0.1 μM IBA within 2 wk. The plants were gradually hardened and established in soil where they flowered and set viable seeds. The regenerated plants were morphologically similar to the field grown plants and showed 100% larvicidal activity against malaria and filarial vectors.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.     

Medium components for shoot cultures of chlorophyll-deficient mutants of petunia inflata

Selected medium components were tested for 30 day growth promotion of shoot tips of Petunia inflata wild type, a cytoplasmic and a nuclear inherited chlorophyll-deficient mutant. Experiments were conducted independently with iron, sucrose, thiamine-HCl, indole-3-acetic acid (IAA), Kinetin (K), 6-benzylaminopurine (BA), coconut milk (CM), casein hydrolysate (CH), and plant extract (PE) an aqueous leaf extract, added to modified Murashige and Skoog (MS) salts and vitamins medium, and pH between 4.0 and 7.0 was also compared. The optimum concentrations of all test components were used to formulate revised MS media especially designed for in vitro shoot growth of chlorophyll-deficient petunia mutants. The optimum medium for the nuclear albino was: MS salts+1.5 mg/l thiamine-HCl+100 mg/l myo-inositol+3.5% sucrose+1% PE+5.0 mg/l IAA+0.3 mg/l K at pH 6.0; for the wild type: MS salts+0.6 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+1.0 mg/l IAA+0.3 mg/l K at pH 5.0 and for the cytoplasmic albino: MS salts+0.4 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+20% CM+3% PE+1.0 mg/l K at pH 5.0. On the revised MS media a 3-, 4- and 5- fold increase in 30 day plant fresh weight occurred for the nuclear, wild type and cytoplasmic chlorophyll-deficient plants, respectively.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.