In vitro multiplication of Peganum harmala--an important medicinal plant

The frequency of shoot regeneration and multiplication of P. harmala was influenced by the type of explant and kind and concentration of hormones. Of the various seedling explants, cotyledonary node exhibited maximum shoot regeneration frequency from axillary region on MS medium supplemented with 5 microM BAP. Addition of 0.1 microM NAA enhanced the efficacy of BAP for multiple shoot regeneration as well as improved the growth of shoots. BAP (5 microM) in combination with NAA (0.1 microM) was found to be the optimal for inducing an average of 4-5 shoots per explant in 75% of the cultures within 5 weeks. Replacement of BAP with other cytokinins at equimolar concentration of BAP i.e. 5 microM was not effective in inducing multiple shoots. Regenerated shoots were rooted on MS medium containing IBA (8 microM) with 80% efficiency. The plantlets were successfully established in soil where 80% of them developed into morphological normal plants.     

Morphogenetic responses of six Philodendron cultivars in vitro

In vitro morphogenic response of nodal explants from six cultivars of Philodendron viz, blue mistic, painted lady, pink prince, pluto, royal queen and green emperor was studied. Frequency and number of shoot formation depend on the cultivars and concentration of BAP. High frequency and number of shoot formation were obtained w hen the nodal explants were cultured in Nitsch medium supplemented with BAP (6.8-11.8 microM), sucrose (2%) and agar (0.45%), initially in the dark for 8-10 weeks followed by 16 hr photoperiod. Regenerated shoots were rooted on medium without growth regulators. After two weeks of hardening, rooted and rootless shoots were established in the soil with more than 90 and 65% survival rates respectively, while the unhardened plantlets showed a much lower percentage (20%) establishment. A standard protocol for the rapid multiplication of Philodendron is presented.     

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10¹⁴ plantlets could be produced in a year from one expiant following the proposed protocol.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

In vitro multiple shoot regeneration and plant production in Alysicarpus rugosus DC. var. heyneanus Baker

A protocol for in vitro multiple shoot regeneration and plant production through seedling (shoot tip) culture was established for Alysicarpus rugosus DC. var. heyneanus Baker. Maximum number of adventitious shoots (14.4) per shoot tip explant were initiated after two subcultures on MS solid medium supplemented with IAA (2.85 microM) plus BAP (2.22 microM) after 4 weeks. Shoot elongation (3.0-3.5 cm) was achieved on MS medium without any hormones. Stunted shoots elongated on half MS medium without growth hormones. Rooting occurred in MS medium containing IAA (1.14 - 2.85 microM) alone or in combination with IBA (0.89 - 2.46 microM) and or NAA (1.07 - 2.69 microM). Maximum rooting was established in MS medium supplemented with IAA (2.85 microM). The plants were acclimatized successfully with 55% survival in pot containing cocoa peat and sand (1:1). After a month, hardened plants were transferred to pots with manure, garden soil and sand (1:2:1) for further growth and finally planted in field.     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

Regeneration of plants from callus tissue of Desmodium affine and Desmodium uncinatum

Plants were in vitro regenerated from leaf callus of Desmodium affine and D. uncinatum. Leaf explants were induced to form callus when aseptically cultured on Murashige and Skoog medium (MS) supplemented with 6 mg dm-3 6-benzylaminopurine (BAP) in combination with 1 mg dm-3 naphthaleneacetic acid (NAA). Regeneration of shoots was induced when callus was cultured on MS medium supplemented with 6 mg dm-3 BAP and 0.01 mg dm-3 NAA. Roots regenerated in high frequency when differentiated shoots were subcultured on MS medium supplemented only with 0.01 mg dm-3 NAA. The regenerated plantlets were successfully grown in pots. Calli from D. incanum failed to regenerate shoots. This revised version was published online in July 2006 with corrections to the Cover Date.     

High Frequency Plant Regeneration from Nodal Explants of Paulownia elongata

Abstract: High frequency of plant regeneration from Paulownia elongata was obtained on Murashige and Skoog (MS) medium and Woody Plant Medium (WPM), with appropriate supplements of growth regulators. Leaves, leaves with petioles, internodes and nodes excised from 3-month-old non-aseptically grown P. elongata were used as explants. The highest shoot regeneration efficiency (93.7 %) was obtained from the nodes of P. elongata on MS medium supplemented with 0.1 mg/ml naphthaleneacetic acid (NAA) and 1 mg/ml 6-benzylaminopurine (BAP). The highest root formation efficiency (100 %) from the regenerated shoots was obtained on WPM supplemented with 1 mg/ml indolebutyric acid (IBA). Rooted plantlets were transplanted to soil with a survival efficiency of almost 100 %. The regeneration system reported here could be useful for rapid multiplication of elite genotypes of P. elongata in a short period of time.     

Micropropagation of squill (Charybdis numidica) through nodule culture

A micropropagation protocol for squill (Charybdis numidica, Hyacinthaceae) was developed using nodule culture. Nodule formation on leaf sections was induced in liquid Murashige and Skoog (MS) medium supplemented with 20 microM N6-benzylaminopurine (BA) under dark conditions. Nodules were cultured on semi-solid MS medium with factorial combinations of BA (0-40 microM) and alpha-naphthaleneacetic acid (NAA) (0-10 microM) under continuous light. Shoot regeneration from nodules occurred at varying degrees on all media. The highest number of shoots was formed on medium containing 2.5 microM NAA and 20 microM BA, while the maximum number of regenerated bulblets per gram nodule was induced on culture medium supplemented with 2.5 microM NAA alone. Regenerated shoots were successfully rooted at approximately 92% on semi-solid MS medium supplemented with 10 microM indole-3-acetic acid (IAA). Plantlets could be hardened and grew well after transfer to the greenhouse. Chemical analyses showed consistent bufadienolide patterns from cloned plantlets and the mother plant.     

In vitro mass multiplication of Ophiorrhiza mungo Linn

A protocol for in vitro mass multiplication of plants through seedling (shoot) cultures was established for Ophiorrhiza mungo. Maximum number of adventitious shoots per shoot culture (10.4 +/- 1.72) was initiated on MS solid medium supplemented with BAP (2.22 microM) after 3 weeks. Shoots were further multiplied (12.8 +/- 2.8) through subculture of intact shoots and reculture of nodal segments of aseptic shoots (6.5 +/- 0.94) in MS solid medium containing BAP (0.89 microM). Shoot elongation (1.27 +/- 0.12 cm) was achieved in the medium containing GA3 (1.44 microM) in two weeks. Rooting was favoured in basal agar medium supplemented with IBA (12.3 microM) plus NAA (1.07 microM). The plants were successfully established (100%) in the pots containing sand and top soil (1:1) mixture in a period of two weeks.     

Micropropagation of Melissa officinalis L. through proliferation of axillary shoots

Multiple shoots were differentiated in cotyledonary nodes of 10 d old seedlings of Melissa officinalis, cultured on MS medium supplemented with BAP (0-4 mg/l). The production of shoots was further induced in subcultures of the original expiant, after the first harvest of shoots (stump), using similar conditions. The highest average number of shoots in the two inoculations was obtained with 2 mg/l of BAP: 24 axillary shoots per explant, 7 in the first inoculation and 17 in the second one. The maximum elongation of shoots was achieved with BAP at 0.2 mg/l, and higher concentrations of the hormone induced a decrease in their size. A range of BAP concentrations between 0.2–0.5 mg/l allowed the production of more shoots with a size suitable for rooting. Roots were induced in 30 d old shoots, transferred to MS medium individually supplemented with IBA or NAA (0–4 mg/l). Micropropagated plants were successfully transferred to soil.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - IDA indole-3--butyric acid - NAA 1-naphthalene acetic acid - FAA formalin-acetic acid-alcohol     

Production of haploids of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.) by anther culture

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.     

Induction of multiple shoots from leaf segments, in vitro-flowering and fruiting of a dwarf tomato (Lycopersicon esculentum)

Multiple shoots were induced from leaf explants of Lycopersicon esculentum cultivar MicroTom, within 20-25d, on MS medium supplemented with 8.9 microM benzylaminopurine (BAP)+1.14 microM indole-3-acetic acid (IAA). For rooting, elongated microshoots were excised and transferred onto MS medium supplemented with 4.9 microM indole-3-butyric acid (IBA). Well-developed roots and flower raceme were obtained on d 7 and 13, respectively, upon transfer of the microshoots onto rooting medium. The flowers self-fertilized in vitro and produced mature fruits in additional 15-17d of culture.     

Somatic Embryogenesis and Plant Regeneration in Kinnow Mandarin

Somatic embryogenesis and plant regeneration from stem explants of kinnow mandarin, a hybrid of king and willow mandarins, are reported. It is an economically important cash crop of India with great deal of production and export. This is for the first time that callus induction and internodal regeneration has been successfully achieved in this citrus cultivar. Callus was induced from nodal segment (1-2 cm) of kinnow mandarin on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and benzyl amino purine (BAP). Cotyledonary stage somatic embryos with regenerated shoots were induced on BAPenriched medium. Roots developed when regenerated shoots were excised and cultured on half strength MS medium supplemented with NAA only.     

Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant

Single medium-based efficient protocols for large-scale multiplication of the rare woody aromatic medicinal plant Rotula aquatica Lour. by means of axillary bud multiplication and indirect organogenesis were established using Murashige and Skoog (MS) medium. There were no significant differences with respect to the induction of shoots per node or callus and roots per shoot on media prepared either with tap water and commercial sugar or those prepared with double distilled water and tissue culture-grade sucrose. The most effective medium for axillary bud proliferation was MS medium fortified with 1.0 mg l(-1 )N(6)-benzylaminopurine (BAP) and 0.5 mg l(-1 )indole-3-butyric acid (IBA), on which shoots were induced at the rate of 15 per node. The excision of node segments from the in vitro-derived shoots and their subsequent culture on medium supplemented with same concentrations of BAP and IBA facilitated enhanced axillary bud proliferation. Callus that developed from the lower cut end of the node explants induced shoots during subculture on half-strength MS medium with 1.0 mg l(-1 )BAP and 0.5 mg l(-1 )kinetin. The shoots developed rooted best on half-strength MS medium supplemented with 0.5 mg l(-1 )naphthaleneacetic acid (NAA). Rooted shoots, following acclimation in the greenhouse, were successfully transferred to field conditions, and 80% of the plantlets survived. When the basal ends of shoots harvested from multiplication medium were dipped in an NAA (0.5 mg l(-1)) solution for 25 days, a mean of 5.6 roots per shoot developed; the transfer to small pots facilitated the survival of 75% of the rooted shoots. Ex vitro rooting by direct transfer of the shoots from the multiplication medium to the greenhouse resulted in a 65% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economically advantageous. About 750 plantlets were procured in a 3-month period starting from a single node explant.     

Micropropagation of sweet orange, Citrus sinensis Osbeck. for the development of nucellar seedlings

Protocol for micropropagation of elite plants of sweet orange (Citrus sinensis) through nucellar embryo culture has been standardized. Three to four nucellar embryos and a zygotic embryo could be excised from a single mature seed and successfully generated as healthy plants in basal MS medium. MS medium supplemented with NAA (1 mg/L) or 2, 4.D (1 mg/L) promoted callus development in both nucellar and zygotic embryos. GA3 (1 mg/L) enriched medium induced plantlets initiation but their growth was very poor. No significant differences were observed between initial growth patterns of nucellar and zygotic seedlings developing from the same ovule. Five to six shoots were obtained from collar region of both category of embryos in MS medium supplemented with BAP (1 mg/L) within 60 days of inoculation. The number of plantlets were almost doubled after their transfer in the same medium and culture for another 30 days. Higher doses of BAP resulted in initiation of callus directly from the embryos. The regenerated shoots (2-3 cm) could be rooted in MS medium supplemented with either only NAA (0.75 mg/L) or NAA (0.50 mg/L) and IBA (2.0 mg/L). A number of plantlets could be obtained from a nucellar embryo grown shoot within a limited time period.     

An improved protocol for micropropagation of elite genotypes of Simmondsia chinensis (Link) Schneider

An efficient micropropagation protocol was developed for elite male and female genotypes of Simmondsia chinensis using nodal segments. Bud initiation was found to be best on Murashige and Skoog’s (MS) medium supplemented with 4.44 μM 6-benzylaminopurine (BAP) and 88.8 μM adenine. Upon sub-culture, 10–15 shoots per explant were obtained when 4.44 μM BAP and 74.0 μM adenine were incorporated in the medium. Increase in KNO₃ concentration in the medium improved shoot multiplication rate and in vitro flowering in 20 % of male cultures. Elongated shoots were harvested, pulse treated for 48 h on liquid medium supplemented with 49.0 μM indole-3-butyric acid, 5.40 μM α-naphthaleneacetic acid and 5.71 μM indole-3-acetic acid for root induction and rooting (92 %) was achieved on hormonal free half-strength MS medium supplemented with 1.37 μM chlorogenic acid, 1 % activated charcoal and 2 % sucrose. After successful hardening, plantlets were transferred to greenhouse with 99 % establishment.     

In vitro propagation of emetic nut Randia dumetorum (Lamb.)

An efficient protocol for in vitro shoot multiplication of Randia dumetorum (Emetic nut) has been developed. The seeds of R. dumetorum were germinated in vitro in MS medium in 5 weeks. Subsequent propagation using shoot tip as an explant was carried out in MS medium along with different concentrations and combinations of BAP (0.5-2.0) and NAA (0.0-2.0). Maximum shoot multiplication was obtained (12.7 shoots per shoot tip) in MS medium containing 1 mg/L BAP and 1 mg/L NAA. Micropropagated shoots were rooted in 1/2 MS medium supplemented with 1 mg/l IBA. This is the first report of in vitro plant propagation of R. dumetorum. In vitro grown plantlets showed a survival rate of 70% after 2 months of transplantation to natural environment.     

Plantlet regeneration from leaf derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andr.

Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP). Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening, with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be produced within eight subcultures from callus obtained from leaf explant through the methods described here.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.