Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

Efficient plant regeneration in <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> Wall., from shoot segment-derived callus

Summary A procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica, through shoot segment-derived callus. Explants used for callus induction were shoot segments derived from 14-d-old axenic plants on Murashige and Skoog (MS) medium supplemented with 15 μM N⁶-benzyladenine (BA). A white friable type of callus was obtained in 4.52 μM 2,4-dichlorophenoxyacetic acid and 2.32 μM kinetin which did not have the potentiality to regenerate. High-frequency shoot differentiation was achieved on transferring the friable callus to MS medium supplemented with 17.8 μM BA and 8.0 μM naphthaleneacetic acid. The highest percentage of calluses forming shoots (65.06±2.26) was achieved in this medium. The organogenetic potential of the regenerating callus was influenced by the age of the culture. Rooting was achieved on the shoots using MS medium with 25 μM indolebutyric acid. The plantlets were acclimatized and established in soil. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Callus induction and plantlet regeneration in Bixa oreliana L., an annatto-yielding tree

Summary A protocol has been developed for plantlet regeneration from seed callus of Bixa orellana L. Seeds demonstrated a high percentage of callus induction (63±7.3%) and a high yield (356±14.7 mg per seed) of white friable callus on Murashige and Skoog (MS) medium containing 5.0 μM l-naphthaleneacetic acid (NAA) and 2.5μM N ⁶-benzyladenine (BA) within 6 wk of culture in the dark. Callus induction frequency was greater under 24h dark as compared to 16h light/8h dark photoperiod or 24h light photoperiod. Increased myo-inositol (MI: 200mgl⁻¹) and addition of ascorbic acid (AA: 200 mgl⁻¹) to the culture medium positively improved callus induction frequency and growth. Shoot differentiation from white friable seed callus was best using 10.0 μM BA and 5.0 μM NAA, where the highest percentage of calluses forming shools (74.9±4.8%), the highest number of shoots per callus (six or seven) and the highest shoot-forming index (5.0) were obtained within 6 wk. Shoots elongated to 4 cm within 4 wk of transfer onto MS medium devoid of growth regulators. Shoots were rooted using half-strength MS medium containing 5.0 μM indole-3-butyric acid (IBA). About 85% of these plants were established in pots containing pure garden soil and organic manure after 3 wk of hardening. Regenerated plants were morphologically uniform with normal leaf, shape and growth patterns. These plants are currently being screened for the presence of agronomically useful genetic variants.     

Influence of type of explant,plant growth regulators,salt composition of basal medium,and light on callogenesis and regeneration in <Emphasis Type="Italic">Passiflora suberosa</Emphasis> L. (Passifloraceae)

Passiflora suberosa is used in popular medicine, improvement programs, and as an ornamental plant. The goal of this study was to establish efficient protocols for plant regeneration and callus induction from nodal, internodal and leaf segments excised from in vitro-grown plants. The different morphogenetic responses were modulated by the type and concentration of plant growth regulators, according to the basal medium and light conditions. Shoot formation occurred through three pathways: (1) development of preexisting meristems, (2) direct organogenesis, and (3) indirect organogenesis. Development of preexisting meristems was observed from nodal segments (1 shoot/explant) in response to α-naphthaleneacetic acid (NAA), picloram (PIC), and 2,4-dichlorophenoxyacetic acid (2,4-D), using two basal media (MS and MSM). Direct organogenesis in this species was obtained for the first time in this work, through shoot development from internodal segments in the presence of 6-benzyladenine (BA). The highest regeneration rates were achieved on MSM medium, regardless of the BA concentration. Indirect organogenesis was achieved from all explant types on media supplemented with BA, used alone or in combination with NAA. The highest regeneration efficiency was obtained from internodal segments cultured on MSM medium plus 44.4 μM BA. Compact, friable, or mucilaginous non-morphogenic calluses were induced by thidiazuron, PIC, 2,4-D, and NAA. High-yielding friable calluses obtained on MSM medium supplemented with 28.9 μM PIC are being used for the establishment of suspension cultures and further analysis of the production of bioactive compounds.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Effect of physical desiccation on plant regeneration efficiency in rice (Oryza sativa L.) variety super basmati

This experiment assessed the effect of partial physical desiccation on plant regeneration efficiency in scutellum-derived embryogenic calluses of rice (Oryza sativa L.) variety Super basmati. A number of callusing cultures were developed, and efficient callus induction was observed on MS (Murashige and Skoog) basal medium supplemented with 2.0 mg/L 2,4-dichlorophenoxy acetic acid. The calluses were proliferated on the same medium for 3 weeks and then shifted to dehydration desiccation treatment for 72 h. The desiccated calluses were cultured on different media for somatic embryogenesis and plant regeneration. A medium with 2.0 mg/L α-napthaleneacetic acid, 10.0 mg/L abscisic acid , 2.0 mg/L kinetin was best for somatic embryogenesis only, but not for further plant development. After 10 d, differentiated calluses were sub-cultured on medium with various concentrations and types of carbohydrates (carbon source) in ¹MS_2j medium. A large number of plantlets (14.51±2.81 and 8.56±2.90 plants/callus) were regenerated via chemical desiccation, on MS with 3% maltose+3% sorbitol and 6% sucrose, respectively. Under dehydration on only simple MS (3% sucrose), 11.23±3.22 plants/callus were developed. Under conditions of dehydration and chemical desiccation, plant regeneration rates were higher than the calluses cultured on simple MS medium in the presence of plant growth regulator. After somatic embryogenesis, >25% plants were sterile. The protocol used here may allow maximum regeneration of normal and fertile plantlets of super basmati rice within 3 months.     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

High frequency androgenesis in indica × Basmati rice hybrids using liquid culture media

An improved procedure has been developed for high frequency androgenesis in indica × Basmati rice hybrids using a liquid culture medium. Anthers from fourteen genotypes comprising of indica × Basmati rice F₁ hybrids, F₂ plants and the parental rice cultivars, were floated in liquid RZM, N6M, and Heh5M media. Anther culture frequencies (percentage of anthers forming calluses) in most of the genotypes were significantly higher in RZM medium (16–75%) compared to those obtained in N6M (7–29%) and Heh5M (7–41%) media. Agarose (1.0% w/v)-solidified MSR1 medium containing 3.0% (w/v) maltose, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹α-naphthalene acetic acid (NAA) induced green shoot regeneration at high frequencies compared to the medium (MSR2) lacking BA. In all the genotypes, microspore calluses initiated in RZM medium regenerated green shoots with over tenfold higher frequencies compared to the calluses initiated in other two media. High plant regeneration frequencies (up to 270 green plants/1000 anthers) were obtained from microspore-derived calluses of some of the F₁ hybrids (Gobind × Basmati 370, Gobind × Taraori Basmati) and F₂ plants (Gobind × Basmati 370, Gobind × Taraori Basmati, HKR86-3 × Taraori Basmati) as compared to their actual parents. Cytological analysis of the root tips of the progeny seedlings of the microspore-derived plants revealed haploids at a frequency of about 50%; 22% of the microspore- derived plants had > 5% spikelet fertility and were diploid. Use of RZM liquid and MSR1 media, respectively for anther culture and plant regeneration resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice F₁ hybrids/F₂ plants which were comparable to those reported for japonica rice varieties/hybrids leading to the improved feasibility of using doubled haploids in genetic, breeding and mapping research with indica rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Long-lasting <Emphasis Type="Italic">Capsicum baccatum</Emphasis> ‘Organogenic callus’ formation

Summary Capsicum regeneration is often obtained through direct (adventious) regeneration and only a few reports claim indirect regeneration (through callus) as an option to obtain complete plants. A possible reason for this may be because Capsicum cultures offer a narrow window in time for the regeneration process to occur, and after that, the ability to regenerate plants rapidly diminishes. In this study, the C. baccatum radicle-side half-seed was the explant of choice to induce the formation of callus on semisolid Murashige and Skoog (1962) medium supplemented with 5 mgl⁻¹ (22.2μM) 6-benzylaminopurine, 1 mgl⁻¹ (5.7μM) indole-3-acetic acid, and 2 mgl⁻¹ (6.1μM) gibberellic acid. Organogenic calluses developed within 4–5 mo. and were subcultured every 2 mo. thereafter. Eventual bud elongation occurred and these shoots developed into complete plants after transfer to medium without plant growth regulators. The organogenic callus type retained its organogenic ability for more than 3 yr.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

In vitro regeneration of Anethum graveolens, antioxidative enzymes during organogenesis and RAPD analysis for clonal fidelity

An efficient in vitro regeneration protocol was developed for medicinally important aromatic plant Anethum graveolens. Nodal segments were cultured onto Murashige and Skoog (MS) basal medium supplemented with different auxins and cytokinins singly as well as in combinations. The optimum callus induction (93.33 %) was obtained on medium fortified with 2.2 μM N⁶-benzyladenine (BA) and 0.21 μM α-naphthaleneacetic acid. The best shoot regeneration (85.7 %) with 12.86 shoots per explant was achieved in two weeks when callus was subcultured on MS medium amended with 2.2 μM BA and 1.85 μM kinetin. The average length of regenerated shoots varied from 3.15 to 4.8 cm. The rooting of regenerated shoots was nearly 100 % on ? MS augmented with 4.9 μM indolebutyric acid with a maximum root length of 5.1 cm. Plantlets were successfully acclimatized with 60 % survival rate. During organogenesis, catalase and ascorbate peroxidase activity increased while superoxid dismutase activity decreased. Clonal fidelity of in vitro raised plants has been checked by random amplified polymorphic DNA using 10 selected decamer primers. It has been found that regenerated plants are true to type plants.     

In vitro propagation of Catalpa ovata G. Don

Plants of C. ovata were regenerated in vitro from shoot tips and nodal explants as well as from cotyledon-derived calluses. For shoot proliferation from shoot tips and nodal segments, Schenk and Hildebrandt (1972) or Lloyd and McCown (1980) basal media, supplemented with 6-benzyladenine (2.2–22.2 μM) alone or in combination with indole-3-acetic acid (0.6 μM), were used. Shoot regeneration through organogenesis was achieved by culturing cotyledons on Schenk and Hildebrandt medium containing indole-3-acetic acid (0.6 μM) and 6-benzyladenine (4.4 μM) or zeatin (22.8 μM). TLC and HPLC analysis showed that the multiple shoots and micropropagated plants exhibited similar iridoid patterns as those of the leaves of original plants of C. ovata. The highest levels of catalpol and catalposide (8.2 and 2.4 % of dry weight, respectively) were found in aerial parts of three-month-old in vitro regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, <Emphasis Type="Italic">Tylophora Indica</Emphasis> (Burm. F.) merrill

Summary Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.     

Efficient callus initiation from leaf of mangrove plant,Bruguiera sexangula in amino acid medium: Effect of NaCl on callus initiation

Experimental conditions for efficient callus initiation from mangrove plants were investigated. As a source explant, leaf ofBruguiera sexangula was used. Mangrove plant is one of the most famous woody plants which can grow at the salty area. The initiated callus can be a suitable material for the investigation of salt tolerant mechanisms of mangrove plants. Leaf pieces cultured in an Amino Acid medium supplemented with 2 μM 2,4-dichlorophenoxyacetic acid and 2 μMN-(2-chloro-4-pyridyl)-N′-phenylurea at 30 C developed calluses. Microscopic observation suggested that the callus was initiated from the tissue in the vascular bundles in the leaf. We also examined the effect of NaCl on callus initiation and short-term culture of the calluses on the leaves. Callus initiation rate decreased with increasing NaCl concentration higher than 100 mM in the culture media. The medium containing 100 mM NaCl produced the largest callus on the leaf, compared with higher or lower concentrations of NaCl.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.