Callus-mediated and direct protocorm-like body formation of Bletilla striata and assessment of clonal fidelity using ISSR markers

Two efficient morphogenetic pathways for micropropagation of Bletilla striata (Thunb.) Reichb. f. have been established through the callus-mediated and direct formation of protocorm-like bodies (PLBs) from protocorms and shoot tips. Green calli were induced from the basal surface of protocorms and the cut-end of shoot tips on Vacin and Went (VW) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene acetic acid (NAA) after 3–5 weeks, with the highest frequency of explants forming callus (48.0 %) from protocorms at 1.0 mg l^?1 2,4-D. The calli obtained from all plant growth regulator (PGR) treatments could proliferate and differentiate PLBs on the PGR-free medium. NAA and 2,4-D significantly enhanced the growth of callus. The fastest growth rate of callus was achieved at the combination of 1.0 mg l^?1 2,4-D and 1.0 mg l^?1 TDZ with 46.2-fold within 3 months. The regeneration of PLBs from callus was significantly improved by 6-benzyladenine (BA), and a mean number of 48.4 PLBs was produced from 100 mg calli at 1.0 mg l^?1 BA within 3 months. BA and thidiazuron (TDZ) promoted the direct formation of PLBs from explants. The highest frequency of direct PLBs formation (76.0 %) and the highest mean number of PLBs per explant (30.2) were observed in protocorms cultured with 0.5 mg l^?1 BA. Assessment of clonal fidelity by inter-simple sequence repeat (ISSR) markers revealed similarity ranges of 99.8–100.0 % between the regenerants and their mother plants and 99.5–100.0 % among the regenerants, which suggested the micropropagation protocols were genetically stable.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

In Vitro Regeneration of Achillea millefolium L from Shoot-tips and Root Segments of Seedlings

This report describes, for the first time, an efficient plant regeneration system for Achillea millefolium L (yarrow), a medicinal plant, via shoot multiplication from shoot-tips and adventitious shoot regeneration from root segments. Higher numbers of shoots were obtained when shoot-tips were cultured on MSMO medium supplemented with 3.0 mg l^?1 BA and 0.5 mg l^?1 IAA, or 5.0 mg l^?1 KIN and 1.0 mg l^?1 IBA, producing 17.3 and 17.0 shoots per explant at 100% frequency, respectively. For adventitous shoot regeneration, only root segments developed shoots when cultured on medium containing a combination of 1 mg l^?1 TDZ, 0.5 mg l^?1 IAA and 0.5 mg l^?1 GA3 (18.9 shoots per explant at 100% frequency), while other types of explants (i.e., cotyledons, leaf lamina and petiole segments) or hormonal combinations tested were found ineffective. Regenerated shoots rooted readily on MSMO medium containing different concentrations of IAA, IBA, NAA or 2,4-D, however, NAA at 0.5 mg l^?1, or IBA at 0.5 or 1.0 mg l^?1 were found to be the most productive. Nearly all of the regenerated plants (98%) survived through the hardening process when the rooted plantlets were kept at 55–65% relative humidity for 2 weeks, which were then planted in pots containing potting soil and kept at 25–35% humidity.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Rapid micropropagation of monopodial orchid hybrid (Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl.) through direct induction of protocorm-like bodies from leaf segments

A competent protocol for accelerated plant regeneration system via direct induction of protocorm-like bodies (PLBs) from leaf of orchid hybrid Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. was developed for the first time to establish a basis for mass production. Using tissue culture technique, the conditions for PLB induction from leaf explants and conversion of PLBs into plantlets were investigated. Leaves were transferred to MS medium containing different types and concentrations of cytokinins (namely, N⁶-benzyladenine, 6-furfurylaminopurine, N-phenyl-N ^??-(1,2,3-thidiazol-5-yl)urea/TDZ and zeatin) for PLB induction. By means of exploring the effects of cytokinins, it was determined that the optimum PLB induction occurred on MS media supplemented with 1.5?mg?l^?1 TDZ; whereby accordingly, PLB induction (with a frequency of 94.8?%) was observed as early as 8?days of culture and an average of 25 PLBs was obtained from 1?cm² leaf segment after 40?days of culture. Variable pressure scanning electron microscopy indicated the different developmental stages of PLBs in detail. Light/stereo microscopic observation showed the maturation of PLBs and gradual formation of shoot and leaf primordia. More than 96?% conversion (with well-developed shoots and roots) was achieved within the next 30?days of culture, when well developed PLBs were transferred in MS medium supplemented with 1?mg?l^?1 BA, 0.5?mg?l^?1 IBA plus 60?mg?l^?1 adenine sulphate. After 60?days of transfer in plastic pots filled with sand and perlite (2:1; v/v) and with charcoal and coconut fibre (1:1; v/v), subsequently, 90?% well-acclimatized plantlets were recovered.     

Genetic transformation and regeneration of transgenic plants from protocorm-like bodies of vanilla (Vanilla planifolia Andrews) using Agrobacterium tumefaciens

An efficient transformation protocol was developed for vanilla (Vanilla planifolia) using protocorm-like bodies (PLBs) derived from shoot tips as explants. Of the ten media tested, Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron (TDZ) produced maximum PLBs per shoot tip. Genetic fidelity of PLB-derived plantlets was confirmed by random amplified polymorphic DNA (RAPD) using 23 random primers. PLBs were co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pBI121 containing the β-glucuronidase (gusA) and neomycin phosphotransferase II (npt II) genes for 3 days in MS medium supplemented with acetosyringone and transferred to selective regeneration medium containing 4.43 μM benzyladenine (BA), 2.68 μM naphthalene acetic acid (NAA) supplemented with 50 mg l^?1kanamycin and 250 mg l^?1 cefotaxime. After 15 days of culture, the surviving explants were transferred to the same regeneration medium but with a higher concentration of kanamycin (75 mg l^?1). Finally, explants surviving after 30 days were subjected to more stringent selection in the regeneration medium supplemented with 100 mg l^?1 kanamycin. Strong β glucuronidase activity was detected in the transformed plantlets by histochemical assay. Integration of T-DNA into the nuclear genome of transgenic plants was confirmed by polymerase chain reaction and Southern hybridization, while expression of transgene was confirmed by northern hybridization. This protocol allows effective and high frequency transformation of vanilla.     

Somatic embryogenesis and plant regeneration in Heracleum candicans Wall

A protocol has been developed for achieving somatic embryogenesis and plant regeneration from petiole-derived callus of Heracleum candicans Wall. Callus was initiated on MS medium supplemented with 0.5 mg l^–1 2,4-D and 0.5 mg l^–1 BAP and subcultured on a medium containing double strength MS macrosalts, 1 mg l^–12,4-D and 0.25 mg l^–1 Kn. Numerous globular embryos were formed on the surface of the callus upon transfer to auxin-rich MS medium that lacked cytokinins. The globular embryos differentiated into mature embryos only when 2,4-D was removed from the medium. Mature embryo formation was significantly influenced by the pH of the medium and the addition of AgNO₃ and ABA. Eighty-five percent of the somatic embryos were converted into plantlets when transferred to a medium supplemented with 0.01 mg l^–1 BAP and 0.01 mg l^–1 IBA. The regenerated plants have been established in soil and appear to be identical to the parent plants in morphology and chromosome number. Received: 5 November 1997 / Revision received: 9 February 1998 / Accepted: 19 February 1998     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Callus Culture and Plant Regeneration from Seedling Explants of Pergularia daemia (Forsk) Chiov

Callus cultures were established from seedling explants of Pergularia daemia (Forsk) Chiov on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins. Optimal callus developed from leaf explants on MS medium supplemented with 2,4-D (2 mg l^?1) + 2iP (0.1 mg l^?1), was used for morphogenesis. Adventitious shoots were regenerated (70%) from the calli on MS medium supplemented with NAA (0.1 mg l^?1)+ BAP (2 mg l^?1). Individual shoots were rooted on half strength MS medium supplemented with 0.1 mg l^?1 IBA. Plantlets with well developed roots were successfully transferred to soil and 50% of the transferred plants survived.     

Somatic embryogenesis and regeneration of Cenchrus ciliaris genotypes from immature inflorescence explants

An efficient, highly reproducible system for plant regeneration via somatic embryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige and Skoog (MS) medium supplemented with 2.0–5.0 mg dm^?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm^?3 N⁶-benzyladenine (BA) for induction of callus. Callus could be successfully induced from all the three explants of both the genotypes. But the high frequency of embryogenic callus could be induced only from immature inflorescence explants. Somatic embryos were formed from nodular, hard and compact embryogenic calli when 2,4-D concentration was gradually reduced and BA concentration increased. Histological studies of somatic embryos indicated the presence of shoot apical meristem with leaf primordia. Ultrastructural details of globular and scutellar somatic embryos further validated successful induction and progression of somatic embryogenesis. Shoots were differentiated upon germination of somatic embryos on MS medium containing 2,4-D (0.25 mg dm^?3) and BA or kinetin (1–5 mg dm^?3). Roots were induced on ½ MS medium containing charcoal (0.8 %), and the regenerated plants transferred to pots and established in the soil showed normal growth and fertility.     

High frequency plant regeneration from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture

An in vitro protocol for efficient plant regeneration has been developed from mature embryo explants of highland barley (Hordeum vulgare L. var. nudum Hk. f.) under endosperm-supported culture. Embryos with (endosperm-supported culture, ES) or without endosperm (non-endosperm-supported culture, NES) were excised from mature seeds and cultured on MS medium supplemented with various concentrations of 2,4-D (1–5 mg l⁻¹) for callus induction. The percentage of callus induction from ES explants was significantly (P < 0.05) lower than that from NES. The highest frequency (97.6%) of callus induction was obtained from NES explants on MS medium containing 3 mg l⁻¹ 2,4-D. When the primary calli were maintained at a reduced concentration of 2,4-D (0.5 mg l⁻¹) for 3 weeks, embryogenic calli were formed. The embryogenic calli were then transferred to MS medium supplemented with different concentrations of BA (1–5 mg l⁻¹) and 500 mg l⁻¹ casein hydrolysate (CH) for shoot regeneration. However, the capacity of plant regeneration from ES explant-derived calli was significantly (P < 0.05) higher than that from NES. The best response (81.3%) was observed from ES explant-derived calli on MS medium containing 2 mg l⁻¹ BA. Regenerated plantlets with well-developed root systems were transferred to pots where they grew well, attained maturity and produced fertile seeds. This method could be employed for genetic manipulation studies.     

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l⁻¹ kinetin and 1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l⁻¹ kinetin, 1 mg l⁻¹ 2,4-D and 100 mg l⁻¹ ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l⁻¹ abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l⁻¹ of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l⁻¹ 6-benzyladenine and 1 mg l⁻¹ α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

High Frequency Shoot Organogenesis in Sorghum bicolor (L) Moench

In vitro morphogenesis that includes enlargement of apical meristems and differentiation of shoot buds on the surface of enlarged meristemoids, have been observed in Sorghum bicolor. The enlargement of apical meristems occurred on the MS medium containing different auxins [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T) and parachlorophenoxyacetic acid (pCPA)] with or without 6-benzylaminopurine (BAP)]. Large number of shoot buds arose over the entire surface of enlarged, green, compact, nodular, hard and shiny meristemoids on transfer to medium containing BAP (2.0 mg l^?1) + (IAA) indole 3-acetic acid (0.5 mg l^-1). Histological observations revealed the origin of shoot apices from the surface of enlarged meristemoids. Efficient rooting was achieved onto half-strength MS medium supplemented with α-napthaleneacetic acid (NAA, 2.0 mg l^?1) and sucrose 2% (w/v). Plantlets were transferred to earthen pots under field conditions for the evalution of several agronomically important characters.     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.