Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Sisal plant regeneration via organogenesis

Callus was initiated from in vitro grown immature leaf and ex vitro grown mature leaf and rhizome explants of Agave sisalana Perr. ex. Engelm, on MS medium containing 2,4-D (9.05 M) and kinetin (4.6 M) or 2,4-D (9.05 M), kinetin (4.6 M) and CH (1000 mg l^–1) or mod. MS (NH₄NO₃, 1500 mg l^–1) containing 2,4-D (9.05 M) and kinetin (4.6 M). Light was essential for callus formation which, however, was different in three types of explants on three different media compositions. Increasing NH₄ ⁺had a negative impact while addition of CH had a positive impact on callus formation. Shoot regeneration from callus from CH-supplemented medium only was achieved for rhizome and immature leaf tissues. The highest rate of regeneration was obtained with BA (26.6 M) as the sole hormone. Shoot buds g^–1 callus varied according to BA concentrations. Shoot proliferation rate increased on half-strength MS medium containing BA (8.9 M). Microshoots developed on MS medium containing BA (2.22 M) and GA₃ (1.44 M) and finally rooted on MS medium containing IAA (11.42 M). Acclimatized rooted plantlets are growing satisfactorily in ex vitro. This is the first report on plant regeneration via organogenesis of A. sisalana.     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Callus induction and plantlet regeneration in ornamental Alocasia micholitziana

Callus induction from petiole explants has been achieved in Alocasia micholitziana `Green Velvet'. The highest percentage (71%) of explants inducing callus was obtained on MS medium supplemented with 0.5 M 2,4-D and 0.5 M kinetin in the dark after 4 months of culture. Shoots were regenerated at the highest frequency of 33.3% under light condition when 0.5 M BA was added to MS medium with the average of 7.8±2.3 shoots per callus explant. The callus-derived shoots rooted on hormone free MS medium and within 4 weeks the plantlets were ready for acclimatization. The regenerated plants appeared morphologically similar to mother plants.     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.)

The growth and differentiation of callus tissues derived from cotyledons of ten cultivars ofCucumis sativus L. were investigated. Cotyledonary explants from all ten cultivars formed callus tissue on Murashige and Skoog (MS) medium supplemented with 0.5 M 2,4-dichlorophenoxyacetic acid and 5 M 6-benzylaminopurine. Fresh weight of the callus tissues averaged 1 to 8 g per flask after five weeks of culture. Shoot development was achieved in three cultivars, Hukchinju, Manchoonchoungjang and Seoul, on MS medium supplemented with 0.5 M -naphthaleneacetic acid and 5 M 6-benzylaminopurine. Reducing the 6-benzylaminopurine concentration to 0.01 M resulted in root formation on callus tissues and on shoots transferred to this medium. All cultivars gave the same response in tests of root formation, but shoot regeneration from callus culture of cucumber cotyledons was dependent on genotype with cultivar Manchoonchoungjang exhibiting the best shoot differentiation capability among the genotypes examined. Examination of mitotic metaphase from the regenerants revealed that all were tetraploid.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

In vitro propagation of Iphigenia indica,an alternative source of colchicine

The present study involves in vitro propagation of Iphigenia indica (Kunth.) through multiplication of whole corms and corm buds. The whole corms produced very small micro-corms, which developed plants individually whereas corm buds multiplied to produce numerous shoots at variable rates in presence of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The best response in corm and bud multiplication was obtained in Murashige and Skoog's basal medium (MS) supplemented with 2.69 M NAA and 8.88 M BAP. The shoots regenerated were further cultured on MS medium containing NAA and indole-3-butyric acid (IBA) for initiation of roots. MS medium with 5.38 M NAA and 4.92 M IBA induced highest percentage of roots (81%) within 2 weeks in culture.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.     

In vitro organogenesis and plant formation in Vigna radiata (L.) Wilczek

In vitro organogenesis was achieved from calluses derived from cotyledon and hypocotyl explants of Vigna radiata on MS medium. Organogenic calluses were induced from both cotyledon and hypocotyl explants excised from 3-day-old seedlings on MS medium containing NAA (1.07 m and BA (2.22 m) and 2,4-D (0.90 m) and BA (2.22 m) combinations respectively. Regeneration of adventitious shoots from cotyledon derived callus was achieved when they were cultured on MS medium supplemented with NAA (1.07 m), BA (8.88 m) and 10% coconut water. Hypocotyl derived calluses produced adventitious shoots when cultured on MS medium fortified with BA (6.66 m), TDZ (2.5 m) and 10% coconut water. Addition of GA at 1.73 m favored maximum 3 elongation of shoots. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 4.90 m IBA. Rooted plantlets, thus developed were hardened and successfully established in field. Among the different carbohydrates and media tested, 87.64 m sucrose and MS+B5 medium proved best for maximum production of shoots. This protocol produced an average of seven plants per hypocotyl derived callus and 15 plants per cotyledon derived callus over a period of 3 months.     

Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effect of explant type, age and plant growth regulators

A callus induction and in vitro plantlet regeneration system for the endangered state flower of Uttaranchal (Saussurea obvallata) was optimized by studying the influence of explant type (root, hypocotyl, cotyledon and leaf), age and different concentrations of plant growth regulators. Explants from 10 to 15-day-old seedlings showed maximum callus induction. Callus formation and shoot differentiation was initiated on Murashige-Skoog (MS) medium containing 6-benzyladenine (BA) and -naphthalene acetic acid (NAA) in all explant types. The best results were obtained using leaf explants: 100% callusing was achieved in MS medium supplemented with 2.5 M BA and 1.0 M NAA, and 100% differentiation along with a multiplication rate of 12 shoots per explant with a combination of 5.0 M BA and 1.0 M NAA. However, the results reflected the existence of high inter-explant variability in response to growth regulators. In vitro rooting of shoots was achieved at an efficiency of 100% in one-half strength MS medium supplemented with 2.5 M indole-3-butyric acid. Application of this protocol has potential for mass multiplication of the target species in a limited time period.     

An efficient <Emphasis Type="Italic">in vitro</Emphasis> method for mass propagation of <Emphasis Type="Italic">Tylophora indica</Emphasis>

A protocol of high frequency shoot organogenesis and plant establishment from stem derived callus has been developed for Tylophora indica (Burm. f.) Merrill. - an endangered medicinal plant. Callus was developed on Murashige and Skoog (MS) medium supplemented with 10 M 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (80 %) of shoot multiplication was achieved on MS medium containing 5.0 M kinetin. The developed shoots rooted best on half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA). The in vitro raised plantlets with well developed shoot and roots were acclimatized successfully and grown in greenhouse.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).