Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Micropropagation of meadowfoam (Limnanthes spp.)

Summary A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N⁶ benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.Abbreviations (BA) N ⁶ -benzyladenine - (IAA) indole-3-acetic acid - (MS) Murashige and Skoog medium, 1962     

Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture

An efficient clonal multiplication system was developed for in vitro propagation of crocin — producing Gardenia jasminoides Ellis plants. Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP 1 mg l^–1) and indole-3-butyric acid (IBA 1 mg l^–1) resulted in multiple shoot initiation at the rate of 21 shoots per explant in 60 d of culture. Transfer of the microshoots into liquid MS medium supplemented with BAP (5 mg l^–1) with two subcultures of 15 d duration in the same medium resulted in 400 ± 25 shoots per explant. Efficient rooting was achieved in MS medium supplemented with -naphthaleneacetic acid (5 mg l^–1). The in vitro raised plants were hardened in a greenhouse and transplanted to the field successfully. The method described will be useful for rapid multiplication of Gardenia for commercial exploitation.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - Kn kinetin - 2ip 6-(,-dimethylallylamino)purine - NAA -naphthalene- acetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid     

Rapid micropropagation for Mucuna pruriens f. pruriens L.

A rapid micropropagation system was developed for Mucuna pruriens f. pruriens using explants from 1-week-old aseptically grown seedlings. Multiple shoot regeneration occurred following an initial callus growth on Revised Tobacco (RT) medium supplemented with 2.7 M NAA and 9.8 M 2iP. Maximum number of shoot regeneration was achieved only from seedling explant 6 to 7 days old. More than 90% of the regenerated shoots could be rooted on half-strength liquid RT medium supplemented with 2.7 M NAA. Plantlets readily adopted to greenhouse conditions. This system provides a new tool for micropropagation of Mucuna pruriens f. pruriens, an important medicinal plant.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - 2iP 2-isopentenyladenine - AdS adenine sulphate - NAA alpha-naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - RT Revised Tobacco medium (Kaul and Staba, 1968)     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Somatic embryogenesis and plant regeneration of squash Cucurbita pepo L cv. YC 60

Plant regeneration from tissue cultures of summer squash, Cucurbita pepo L., cv. YC60, has been observed. Somatic embryos organized from shoot apex derived callus cultured on Murashige and Skoog (MS) medium supplemented with 1.2 mg/l 2,4,5-trichlorophenoxyacetic acid, 0.8 mg/l benzylaminopurine, and 0.1 mg/l kinetin. Embryos developed into plantlets by transfer of immature somatic embryos to MS medium with 0.05 mg/l NAA and 0.05 mg/l kinetin. Regenerated plants appeared morphologically normal and set fruits with seeds which could germinate normally.Abbreviation BAP 6-benzylaminopurine - 2,4-D 2, 4dichlorophenoxyacetic acid - IAA indole-3-acetic acid - KN kinetin - NAA -naphthyleneacetic acid - MS Murashige and Skoog - 2,4,5-T 2, 4,5-trichlorophenoxyacetic acid     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts

An efficient method was developed using floating membrane rafts (Liferaft) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.Abbreviations BA 6-benzylaminopurine - GA₃ Gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid     

Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid     

Plant regeneration and ploidy variation in culture derived plants of Asclepias curassavica L

Clonal propagation of medicinal milkweed, Asclepias curassavica L. (Asclepiadaceae) was achieved by culturing excised nodes on MS medium (Murashige and Skoog, 1962) supplemented with different hormone combinations. Both BAP and Kn were found equally effective for shoot initiation. IAA and NAA were found suitable for root induction. Combinations of Kn and NAA induced both roots and shoots after 30 days of culture. Chromosomal variation was observed in the roots of in vitro regenerated plants. Regenerants with higher chromosome number (33; 2n=22) obtained on MS medium in response to 9.2 M Kn+10.7 M NAA showed vigorous growth and higher propagation rates in culture than the plants possessing less than the diploid chromosome number (2n–2=20, 2n–4=18). Such variations are more likely due to genetic fitness of different aneuploids grown on a particular nutrient medium.Abbreviations IAA 3-Indoleacetic acid - NAA Naphaleneacetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - IBA Indolebutyric acid - Kn Kinetin - BAP Benzyladenine - GA₃ Gibberellic acid - S Shoot - R Root     

Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.)

Summary Sunflower hypocotyl protoplasts (Helianthus annuus L.) from 5 PIONEER genotypes (PT024, SMF3, EMIL, HA300*PT024, VK5F) and 1 public line (RHa 274) formed colonies at frequencies of up to 60% when plated in 0.25ml agarose beads in a modified L4 medium (Lenée and Chupeau 1986) containing 3mg/l NAA, 1mg/l BA and 0.1mg/l 2,4-D, and 1000mg/l casamino acids. Protoplast-derived colonies grew slowly into calli. Organogenesis was obtained from callus of PT024 on a MS medium containing NAA and BA at 1mg/l and GA at 0.1mg/l. Freshly excised shoots were induced to root by an IAA treatment. Regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.Abbreviations BA 6-benzylaminopurine - NAA -naphtaleneacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog mineral elements - B5 Gamborg mineral elements     

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.Abbreviations BAP 6-benzylaminopurine - Dd H₂O Double glass distilled water - IAA indole-3-acetic acid - IBA indole-3-butyric acid - K Kinetin - MS Murashige and Skoog's (1962) medium - RH Relative humidity CIMAP Publication No. 876     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Morphogenetic responses of cultured cells of cambial origin of a mature tree — Dalbergia sissoo Roxb

Regeneration of plantlets was achieved from cell suspension derived calli of cambial origin from mature elite trees of Dalbergia sissoo. Callus proliferation occurred on the cambial tissue pieces cultured on MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (2.0 mg/1) and benzylaminopurine (0.1 mg/l). Suspension cultures were obtained by transferring and agitating callus lumps in liquid medium composed as above. Aggregates of about 30 cells were plated on semi solid medium, which developed into calli. Shoot bud differentiation was observed in the calli transferred to medium devoid of auxin but containing 0.5–2.0 mg/1 benzylaminopurine. The isolated microshoots were rooted on modified MS medium containing low organic salts and auxins.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IPA indole-3-propionic acid - KN kinetin - MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

Clonal propagation of Acacia catechu Willd. by shoot tip culture

A method is described for in vitromicropropagation through shoot apices of Acaciacatechu Willd., a semi-arid tree valued for Katha (atanin-like substance obtained from red heart wood of10–20 year old trees) and timber. Explants wereexcised from 15-days-old in vitro grownseedlings raised from superior seed stocks. Shoot budinduction from shoot apex explants was observed onMurashige and Skoog's (MS) [12] medium containingvarious growth regulators. A maximum of 12 shoots wasobtained on MS medium supplemented with 1.5 mg/l 6-benzylaminopurine (BAP) and 1.5 mg/l kinetin.Well-developed shoots (3–4 cm long) were rooted on strength MS medium with 3.0 mg/l indole-3-acetic acid (IAA) and sucrose 1.5%. In vitro regenerated plantlets of A. catechu were transferred to field conditions.