In vitro morphogenetic responses from cotyledonary explants of immature zygotic embryos of Pterocarpus santalinus

Plant Cell, Tissue and Organ Culture (PCTOC) - Pterocarpus santalinus is an endangered species with high therapeutic and commercial value that needs urgent protection. The study developed an easy...     

In vitro morphogenetic studies on three apomictic species of Garcinia

Comparison of morphogenetic potential of three important Indian species of Garcinia??G. indica, G. cambogia and G. xanthochymus has been reported. Apomictic seeds of G. indica were found to be morphogenetically most potential followed by G. cambogia. The explants of G. xanthochymus were highly recalcitrant towards in vitro conditions and failed to induce adventitious buds on any of the media tested. High frequency direct shoot bud differentiation was induced in aseptic seed cultures of G. indica and G. cambogia on MS medium supplemented with cytokinins (BAP, kinetin or TDZ). Amongst the three cytokinins tested, TDZ (0.1?C0.5???M) was most effective for adventitious bud differentiation in both G. indica and G. cambogia, however, the proliferating buds failed to elongate. Substantial number of buds induced on BAP supplemented media elongated into shoots after subculture on elongation medium. Addition of NAA along with cytokinins in the induction medium enhanced callusing without improvement in bud induction response. The induced adventitious buds were elongated on MS basal medium containing 0.2% activated charcoal. Direct rooting was achieved in both G. indica and G. cambogia on auxin supplemented media with best response at 10???M IBA concentration in both the species. The in vitro raised plantlets showed 90% survival in the field when transferred after hardening and acclimatization.     

In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants

Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1-naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-buds proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l: mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 ∶ 5.0, 0.0 ∶ 5.0, and 0.1 ∶ 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explants and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/l IAA or 0.4 mg/l NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.     

In vitro regeneration of apomictic bluestem grasses

Explants from immature inflorescences of four genotypes of Old World bluestem grasses, (Bothriochloa spp.), produced callus tissue on Linsmaier and Skoog (RM) and 1/2 Murashige and Skoog (1/2 MS) media containing high levels of growth regulators. Callus masses were composed of two distinct tissue types, one a compact, white, embryogenic portion (E calli), the other soft, translucent, gelatinous and nonembryogenic (NE calli). When transferred to medium with a reduced level of 2,4-D, and/or supplemented with zeatin, E callus underwent further organization culminating in shoot production. Light and scanning electron microscopy confirmed the embryogenic pathway of differentiation. Genotype significantly affected callus induction frequency and the number of plants regenerated. The RM medium induced more explants to initiate callus compared to the 1/2 MS medium. Age of the inflorescence explant, as indicated by size, was critical for callus induction. Inflorescences with racemes 8 mm in length were superior to older ones. Five-hundred-twenty-two plantlets were regenerated and grown to maturity.     

In vitro propagation of Cleome spinosa (Capparaceae) using explants from nursery-grown seedlings and axenic plants

Summary Two independent experiments were performed to establish micropropagation of Cleome spinosa from stem segments. In the first experiment, direct shoot organogenesis on hypocotyl explants from 2-mo.-old nursery-grown seedlings was obtained on Murashige and Skoog medium with different combinations of benzyladenine (BA) and 6-furfurylaminopurine, added either individually or in combination. Best proliferation rates occurred in the presence of 2.2 and 4.4 μM BA and the highest mean number of shoots was produced in response to 4.4 μM BA. In the second experiment, regeneration via direct organogenesis was also obtained from nodal and internodal segments of axenic plants cultured in the presence of BA (4.4 and 8.8 μM) in association with indole-3-acetic acid (IAA) (0.57 and 1.14 μM). Internodal explants were the most responsive on all media tested. The best mean number of shoots per explant was achieved on medium with 4.4 μM BA in association with 0.57 μM IAA. Histological studies of the globular structures formed at the apical portion of the explants revealed direct shoot regeneration and adventitious shoot differentiation from meristematic centers around the vascular bundles of the primary regenerants. All shoots elongated and rooted on MS0 medium. The acclimatization rates ranged between 70 and 84%. Plants reached to maturity and flowered 4 mo. after transfer to ex vitro conditions.     

In vitro morphogenesis from excised leaf explants of Digitalis obscura L.

The morphogenic capacity of Digitalis obscura leaf explants cultured in vitro has been studied, noting factors promoting the differentiation of roots, buds and shoots as well as those promoting callus proliferation. Complete plant regeneration was obtained only by first culturing the leaf explants in a medium with NAA and BA to induce formation of buds, and subsequently transferring them to a medium without growth regulators to achieve the further development of shoots.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

In vitro regeneration of Sophora toromiro from seedling explants

Embryonic axes with cotyledons, shoot-tips of embryonic axes, isolated cotyledons, as well as axillary buds and leaves from 20-year-old trees of Sophora toromiro, were evaluated for their capacity to trigger organogenesis and to regenerate plantlets under in vitro conditions. Embryonic shoot-tips were the only explants capable of regenerating plants. They developed rapidly in vitro in the presence of NAA and BA while in subculture roots were induced at the proximal end in the presence of 0.49 μM IBA within 40–60 days. Development was completed with a subculture phase under non-sterile conditions using a mixture of equal parts of sterilized vermiculite/sand/soil in growth chambers, before final acclimation in the greenhouse. In the presence of NAA, BA and GA₃, whole embryonic axes formed multiple shoots that branched when grown in 2.27 or 11.35 μM TDZ in subculture. Similarly, callus was initiated at the embryo axis base, developing into several new shoots in the presence of TDZ. Because of the relatively high shoot induction rate along the embryonic axis, this axis presents a valuable source of new juvenile explants. Growth and rhizogenesis was satisfactory only when organs from seed pods of the year or from the previous season were used. Experiments with isolated cotyledons produced callus only, while axillary buds and leaves did not show any responses in the presence of several growth regulators assayed. Inoculation of seedlings with various strains of rhizobia under in vitro conditions resulted in root outgrowths, but not in nodules that are typical of rhizobia infection. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro tetraploid induction from leaf explants of Populus pseudo-simonii Kitag.

Tetraploid plants were produced from leaf explants of diploid Populus pseudo-simonii by treating the leaves with colchicine. Leaf explants were cultured on MS basal medium containing 1.78 μM BA and 1.08 μM NAA for 0, 6 and 12 days, and then transferred to the same MS liquid medium with colchicine at concentrations of 25, 50 and 75 μM for 1, 2 and 3 days. The highest efficiency of tetraploid induction was 14.6% by treating leaf explants that were pre-cultured for 6 days and then cultured in liquid MS with 50 μM colchicine for 3 days. Flow cytometric analysis was used to screen the tetraploids out from the regenerated plants and chromosome number counting was employed to confirm the polyploidy level. Size and frequency of leaf stomata between diploid and tetraploid plants were demonstrated to have significant differences.     

In vitro regeneration of Semecarpus anacardium L. from axenic seedling-derived nodal explants

Semecarpus anacardium (Anacardiaceae), a deciduous forest tree, is a potent source of medicinal compounds. Poor seed viability of this species limits the conventional propagation practice. Proliferation of shoots from axillary meristem was achieved in semisolid WPM medium supplemented with BAP 4.44 μM and KN 4.64 μM. Factors including culture vessels, gelling agents and antioxidants were identified and optimized for proliferation and growth of shoots in vitro. Cotton-plugged culture vessels were more favorable. Phytagel 0.2% as gelling agent and activated charcoal 0.2% as antioxidant were superior to other agents and antioxidants tested. All the shoots rooted in half-strength WPM liquid medium with IBA 2.46 μM. Rooted shoots survived (91%) in the soil–sand 1:1 mixture. Ex vitro rooting of shoots and hardening of plants were achieved in 80% of the explants in the soil–sand mixture. Hardened plants were maintained in a greenhouse. This is the first report on in vitro regeneration of Semecarpus anacardium.     

Direct shoot regeneration from excised leaf explants of in vitro grown seedlings of Alstroemeria L.

A two-step protocol for the induction of shoots from Alstroemeria leaf explants has been developed. Leaf explants with stem node tissue attached were incubated on shoot induction medium for 10 days, and then transferred to regeneration medium. Shoots from the area adjacent to the region between the leaf base and node tissue regenerated within 3 weeks after transfer to the regeneration medium, without a callus phase. The best induction was obtained with Murashige and Skoog medium containing 10 μm thidiazuron and 0.5 μm indole butyric acid. The regeneration medium contained 2.2 μm 6-benzylaminopurine. After several subcultures of the leaf explants with induced shoots, normal plantlets with rhizome were formed. In Alstroemeria, the percentage of responding leaf explants is more important than the number of shoots regenerated per leaf explant, because rhizome formation is the most important factor for micropropagation. The effect of other compounds in the induction medium, including glucose, sucrose, silver nitrate, and ancymidol, on regeneration was also investigated. Received: 14 June 1996 / Revision received: 27 September 1996 / Accepted: 20 October 1996     

In vitro organogenesis from leaf explants of Annona squamosa Linn

Multiple shoot formation was induced from excised leaf explants of Annona squamosa Linn. (custard apple) seedlings on a Murashige and Skoog basal medium containing benzylaminopurine and kinetin. Various auxins in combination with the above medium produced callusing of the explants. In an investigation of environmental factors affecting shoot induction it was seen that the maximum number of shoots were obtained using the leaf base with petiole at a temperature of 27°C and a light intensity of 1000 lux. Roots were initiated erratically when individual shoots were treated with an auxin and then transferred to an auxin free medium. The process of the development of adventitious buds in leaf culture was analysed histologically.NCL Communication No. 3104.     

In vitro regeneration of Alnus cremastogyne Burk from epicotyl explants

Summary Multiple shoots were grown from seedling explants of Alnus cremastogyne Burk by a two-stage culture procedure: initiation on WP medium supplemented with 2–8 M benzylammopurine(BAP) for 6 weeks, thereafter 3 weeks of subculture(shoot multiplication) on the same medium with 1 M BAP. A 5–9 fold multiplication rate was achieved. Type and concentration of sugar used in the multiplication medium were shown to be critical factors for both multiple shoot induction and bud elongation, the optima being 87.5mM glucose and 87.5mM sucrose respectively. After transfer to half-strength WP media either containing indolebutyric acid (IBA) or lacking plant growth regulator, almost all the shoots rooted. However, high rhizogenesis could be achieved only with shoots cultured in rooting medium containing 87.5mM sucrose or 175mM glucose, and shoots from multiplication media containing 87.5mM sucrose. Survival of the plantlets following transfer to vermiculite was 100%.Abbreviations BAP 6-benzylaminopurine - 2iP N⁶-(²-isopentenyl)adenine - kinetin 6-furfurylaminopurine - zeatin trans-6-(4-hydroxy-3-methylbut-2-enyl)aminopurine - IBA indol-3-butyric acid - WPM Woody plant medium (Lloyd and McCown, 1981)     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

In vitro plantlet regeneration from embryonic explants ofPinus pinea L

Summary Clonal propagation ofPinus pinea L. was achieved by organogenesis on cotyledon explants and the influence of several factors on adventitious bud production and development was investigated. Gupta and Durzan (DCR) medium with benzyladenine (5 μM) induced higher bud production. Bud development and shoot elongation required subcultures on medium with activated charcoal. Rooting was obtained after 10 d culture on medium containing IBA (10 μM).     

In vitro organogenesis from leaf explants of Annona squamosa Linn

Multiple shoot formation was induced from excised leaf explants of Annona squamosa Linn. (custard apple) seedlings on a Murashige and Skoog basal medium containing benzylaminopurine and kinetin. Various auxins in combination with the above medium produced callusing of the explants. In an investigation of environmental factors affecting shoot induction it was seen that the maximum number of shoots were obtained using the leaf base with petiole at a temperature of 27°C and a light intensity of 1000 lux. Roots were initiated erratically when individual shoots were treated with an auxin and then transferred to an auxin free medium. The process of the development of adventitious buds in leaf culture was analysed histologically.     

In vitro plant regeneration from leaf explants of Ophiorrhiza japonica

An efficient in vitro plant regeneration system from leaves of Ophiorrhiza japonica Blume was established for the first time. Callus formation rate was more than 90.4 % from leaf segments on Murashige and Skoog (MS) supplemented with either α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA). The highest shoot regeneration (78.9 %) was achieved on MS medium containing 2.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA, with an average of 9.4 shoots developed per leaf segment. Shoot regeneration was also improved when the leaf explants were cultured in MS basal medium supplemented with 0.5 % (m/v) polyvinylpyrrolidone (PVP). The leaf explants from seedlings with age of about 18–27 d showed the highest shoot regeneration. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA), which averagely produced 24.8 roots per shoot. The plantlets were transferred to soil, where 100 % survived after 1 month of acclimatization.     

In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L.

Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to be highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminopurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation.Abbreviations MS Murashige and Skoog - IAA 3-indoleacetic acid - BA 6-benzylaminopurine     

In vitro organogenesis and somatic embryogenesis from leaf explants of Leucosceptrum canum sm.

Plantlets were obtained from leaf explants of a Labiatae tree — Leucosceptrum canum Sm. using plant tissue culture techniques. Two types of calli proliferated from the leaf explants when grown on different media, one of which was amenable to somatic embryogenesis. Differentiation of the embryoids started from the fourth passage of culture and continued up to the seventh passage. The number of embryoids decreased with the age of the callus. The capacity of such embryoids to form entire plantlets was studied using different nutrient mileux. Embryoids formed plantlets on Murashige and Skoog's (MS) medium fortified with benzylaminopurine plus indolebutyric acid. Organogenesis was observed in shoot-buds derived from explants of in vitro regenerated plantlets on MS basal medium supplemented with benzylaminopurine. Culture regenerated plantlets were transferred to MS medium without sucrose and growth hormones; finally transferred to pots containing sterile vermiculite where they are growing.Abbreviations MS Murashige and Skoog's medium - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - Kn kinetin - BAP benzylaminopurine - CW coconut water     

In vitro plant regeneration from cotyledon explants of Swainsona salsula Taubert

An effective protocol has been developed for plant regeneration from cotyledon explants of Swainsona salsula Taubert (Saline swainsona), a medicinal and agronomic shrub. Adventitious shoots were obtained from 83.2% of cotyledon explants from 3-day seedlings cultured on Murashige and Skoog (MS) medium containing 2.0 mg l⁻¹ thidiazuron (TDZ), with an average of 9.3 shoots per explant. Individual elongated shoots were rooted on half strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid (IBA), with 59.3% success. Regenerated plants with well developed shoots and roots were successfully transferred to soil, without detectable variants. Histological observation revealed that shoots developed from cotyledon explants via organogenesis, with little callus. This revised version was published online in June 2006 with corrections to the Cover Date.