TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

Plant regeneration from different explant types of Bituminaria bituminosa and furanocoumarin content along plant regeneration stages

The first protocol for in vitro plant regeneration from different explants of Bituminaria bituminosa, a pasture and medicinal species, has been established. Three explant types (petiole, leaflet and petiole-leaflet attachment “PLA”) cultured on media with different combinations of benzylaminopurine (BA; 5.0, 10.0 or 20.0 μM) and naphthalene acetic acid (NAA) or indole acetic acid (IAA; 0.5 or 5.0 μM) were tested for calli induction, and with 5 μM BA + 0.5 μM NAA or IAA for shoot development. The average number of shoots (≥5 mm) per callus depended on the explant type and the calli induction medium. The highest average number of shoots per callus was achieved by culturing leaflet and PLA explants on 5 μM IAA + 10 μM BA for calli induction and on 0.5 μM IAA + 5 μM BA for shoot development, and by culturing petiole explants on 0.5 μM NAA + 10 μM BA followed by a second culture on 0.5 μM NAA + 5 μM BA. The highest frequency of shoot rooting was achieved with 10.0 μM NAA and 1.0 μM gibberellic acid (GA₃). Rooted plants were acclimatised in a culture chamber, reaching 96 % survival. Acclimatised plants were transferred to a greenhouse and finally to the field, reaching 100 % survival. The furanocoumarin (FC) accumulation was evaluated in organogenic calli, in vitro shoots, ex vitro plants in the greenhouse and in ex vitro plants in the field (after 1 and 4 months of acclimatisation). The content of FCs depended on the plant material evaluated, being higher in ex vitro plants in the field (up to 9,824 μg g^?1 DW total FC) and lowest in organogenic calli (up to 50 μg g^?1 DW total FC). This effect may be due to cell organization, longer exposure to environmental factors and the developmental stage.     

Cardenolide estimation in callus-mediated regenerants of Digitalis lamarckii Ivanina (dwarf foxglove)

Digitalis cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 μM) and α-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 μM). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 μM BA and 10.8 μM NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 μM) and/or gibberellic acid (0.7 or 1.4 μM) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 μM) or NAA (1.3 to 5.2 μM), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their cardenolide contents. Lamina extracts showed nearly three times higher cardenolide accumulation than petiole extracts. Of the cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain cardenolides from D. lamarckii.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

High-frequency shoot regeneration from leaf explants through organogenesis in bitter melon (<Emphasis Type="Italic">Momordica charantia</Emphasis> L.)

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro (15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l⁻¹ sucrose, 2.2 g l⁻¹ Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO₃). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA₃). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an average of 40 plants per leaf explant with a culture period of 98 days.     

Evaluation of regeneration potential of mature embryo derived callus in Indian cultivars of barley (Hordeum vulgare L.)

A protocol for plant regeneration in Indian cultivars of barley (Hordeum vulgare L.) has been developed using mature embryo culture. The influence of various auxins 2,4-D (2,4-dichlorophenoxyacetic acid), Dicamba (3,6-dichloro-o-anisic acid) and Picloram (4-amino-3,5,6-trichloropicolinic acid) on the callus induction and subsequent plant regeneration revealed highest percent of callus induction form cultivar (cv) BL 2 on MSB₅ medium (MS salts + B₅ vitamins) supplemented with 6 mg l^?1 Picloram, but maximum number of shoot buds (6–13) were regenerated on MSB₅ medium containing 0.5 mg l^?1 Picloram. Regenerated shoots were rooted on half-strength MSB₅ medium. Plantlets were successfully transferred to soil and grown to maturity in greenhouse. The effect of copper sulphate revealed significant improvement in callus induction and plant regeneration when the concentration of CuSO₄ was increased to 3 μM (30 times higher than normal MS medium) for cv BL 2. Regeneration potential differed for different cultivars of barley used, with highest for cv BL 2 and lowest for cv BH 924. We conclude that the Indian barley genotypes exhibit plant regeneration from mature embryo cultures. The protocol has potential application in barley improvement through genetic engineering.     

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful immature embryo culture and efficient plant direct regeneration via organogenesis from cotyledons, which showed different patterns, was established for the “Xuemei” cultivar of Prunus mume. For immature embryo culture, high frequency plantlet forming (89.5%) from embryo axis was obtained on half-strength Murashige and Skoog (½MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). At the same time, shoots direct differentiation from cotyledons with the embryo axis development was also observed on ½MS medium containing 2.2 μM BA together with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when embryo axes were removed from cotyledons and cultured on ½MS medium supplement with 13.2 μM BA, 2.7 μM NAA (72.9%) or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA (84.2%), respectively. Regenerated shoots were successfully rooted on ½MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of embryo axes, BA and TDZ, on cotyledons’ regeneration were investigated in detail. The rooted plantlets were transferred to soil successfully with normal morphology.     

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

In vitro shoot growth, direct organogenesis and somatic embryogenesis promoted by silver nitrate in Coffea dewevrei

Direct differentiation of shoot buds in Coffea dewevrei was evident from the seedling shoots with collar region and also from collar region end of hypocotyl segments in presence of 40 μM AgNO₃, 8.88 μM of BA and 2.85 μM of IAA. Apart from this, shoot end of hypocotyl explants mainly supported yellow friable callus or somatic embryos. Subsequent transfer to the same medium induced secondary somatic embryogenesis. The collar region of the hypocotyl explants not only showed direct organogenesis by producing 1–3 shoots per explant and also able to produce globular somatic embryos and embryogenic yellow friable callus. Similarly direct somatic embryogenesis along with yellow friable embryogenic callus formation on 1/2 strength MS medium comprising 1.47 μM IAA, 2.22 μM BA and 40 μM AgNO₃ was noticed from cut portion of in vitro leaf and stalk of regenerated plants. The microshoots rooted well upon subculturing onto the same medium in 6 weeks and showed 60 % survival in green house and resumed growth upon hardening.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Direct regeneration from in vitro leaf and petiole tissues of <Emphasis Type="Italic">Populus tremula</Emphasis> ‘Erecta’

Dormant buds from a mature tree of Populus tremula ‘Erecta’ were incubated on a Murashige and Skoog (MS) medium supplemented with 1.0 μM thidiazuron (TDZ). Induced shoots were then proliferated on medium of MS or Woody Plant Medium (WPM), or Driver and Kuniyuki Walnut (DKW) supplemented with varying levels of benzyladenine (BA). Overall, shoots grown on MS medium supplemented with 1.25–2.5 μM BA exhibited the highest frequency of shoot proliferation (>95%) and more than 60% of responding explants produced more than five shoots per explant. Shoot organogenesis was induced from both leaf and petiole explants incubated on WPM medium containing BA, or TDZ, or zeatin. Among the different cytokinins tested, zeatin induced the highest frequency (average 72.1%) of shoot organogenesis. None of explants survived on media containing no cytokinins within 6–8 weeks following culture. Overall, a higher frequency of shoot regeneration was obtained from petioles than from leaf explants. The highest frequency of regeneration was achieved when petioles were incubated on WPM containing 10–20 μM zeatin. Addition of naphthaleneacetic acid (NAA) did not have a significant effect on shoot regeneration in all treatments. Shoot organogenesis was directly induced from petiole explants without intervening callus. Regenerated shoots were easily rooted on all tested media supplemented with 0.5 μM NAA. Rooted plants were transferred to potting mix and grown in the greenhouse.     

In vitro propagation of Agave fourcroydes Lem. (Henequen)

In vitro plant regeneration of Agave fourcroydes Lem. (Agavaceae) is described. Results suggest that the NO₃ ^-:NH₄ ⁺ balance in the culture medium is a key factor controlling callus growth and organogenesis in rhizome cultures. Stem callus showed limited organogenic capacity, but high cytokinin concentrations induced adventitious shoot formation on stem explants. When these shoots were excised and subcultured, new callus formed at their base from which new shoots arose. The shoots from stem explants and rhizome callus formed extensive root systems in vitro and were transferred to pot culture with a 90% survival rate.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Salvia nemorosa</Emphasis> L. from shoot tips and leaf explants

Summary Shoot tips and leaves excised from in vitro shoot cultures of Salvia nemorosa were evaluated for their organogenic capacity under in vitro conditions. The best shoot proliferation from shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with 8.9 μM 6-benzylaminopurine (BA) and 2.9 μM indole-3-acetic acid (IAA). Leaf lamina and petiole explants formed shoots through organogenesis via callus stage and/or directly from explant tissue. The highest values for shoot regeneration were obtained with 0.9 μM BA and 2.9 μM IAA for lamina explants. No shoot organogenesis was obtained on leaf explants cultured on MS medium supplemented with α-naphthaleneacetic acid (NAA). The regenerated shoots rooted the best on MS medium containing 0.6 μM IAA or 0.5 μM NAA. In vitro-propagated plants were transferred to soil with a survival rate of 85% after 3 mo.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Plant regeneration from callus cultures of Psoralea corylifolia Linn

Plantlet regeneration via organogenesis was achieved in callus cultures derived form mature leaves, stems and leaves, petioles and roots of young seedling of Psoralea corylifolia on Murashige and Skoog medium supplemented with 2.5–3.0 mg L^-1 BA, 1.0 mg L^-1 NAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of hormones in the nutrient media. Shoot buds regenerated more readily from juvenile explants (seedling source) as compared to the mature explants. Addition of adenine sulphate (5 mg L^-1) to the culture medium increased the growth of shoot buds. Optimum responses were obtained in hypocotyl and leaf explants using NAA in combination with BA, the highest rate of shoot bud regeneration being in hypocotyl explants. Rooting was readily achieved on the differentiated shoots on MS basal media without growth regulators. Regenerated plantlets were successfully established in the greenhouse.     

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

Abstract

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36?h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0?μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0?μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5?μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15?μM TDZ for 24?h) explants was achieved on WPM augmented with 6.0?μM TDZ and 0.5?μM NAA after 8?weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (～0.5?g) and were transferred onto WPM fortified with different concentrations (2.0–9.0?μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0?μM) and gibberellic acid (GA_3, 0.5–3.0?μM). Maximum shoot number (10.4?±?0.74) and average shoot length (4.75?±?0.71?cm) were observed in WPM enriched with 2.0?μM BAP, 1.0?μM IAA and 1.5?μM GA₃ after 8?weeks of incubation. The developed shoots (4?cm) were excised, pulse-treated for 24?h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0?μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60?days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.     

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

Abscisic acid promotes shoot regeneration in Arabidopsis zygotic embryo explants

An efficient shoot organogenesis protocol for Arabidopsis zygotic embryo explants of Landsberg erecta ecotype was established. This de novo shoot organogenesis protocol has three different steps, i.e., induction of callus in an auxin-rich callus induction medium, the formation of green-organogenic callus in the shoot induction medium (SIM), and the final morphological differentiation of shoot in the hormone-free shoot development medium (SDM). Abscisic acid (ABA), auxin, and cytokinin (CK) were used in the SIM. Individual plant growth regulators as well as their combination were studied to understand their importance in the shoot induction treatment. We found that a combination of ABA + CK and ABA + CK + auxin induced higher shoot organogenic ability in the callus than ABA, CK, and auxin alone. Optimum ABA concentration on shoot organogenesis was determined to be 10^?5 M. Morphological characterization of callus induction and shoot organogenesis events indicated that calli were derived from the cotyledons of zygotic embryo explants and the formation of green organogenic calli was specific to the exogenous inclusion of ABA + CK in the SIM. During the time of shoot development, the green organogenic callus became darker green due to the formation of anthocyanins. Shoot organogenic calli in the SIM and the SDM were easily identified by the green-colored calli and anthocyanin pigments, respectively. Furthermore, we demonstrated the significance of exogenous and endogenous ABA in shoot organogenesis by fluridone treatments. The inclusion of ABA in SIM has a significant effect on shoot formation.