Effect of water stress mediated through agar on <Emphasis Type="Italic">in vitro</Emphasis> growth of potato

With the objective to develop a practical method of screening potato for drought tolerance, shoot and root growth in plantlets raised in vitro (from nodal cuttings drawn from in vivo as well as in vitro grown plantlets) were studied in three genotypes with known root mass production under field conditions. Different levels of water stress were induced using five concentrations of agar in MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium. Water potential of various media ranged from −0.70 MPa to −0.98 MPa. Water stress in culture adversely affected plantlet growth, and the responses varied with genotype and explant source. Genotype IWA-1 was less affected than Konafubuki and Norin-1. In the experiment with explants from in vivo grown plants, the time to rooting was considerably delayed in Konafubuki and Norin-1 by an increase in agar concentration, but no such effect was observed in IWA-1. In all media, the mean number of roots and root length was greater in IWA-1 than Konafubuki and Norin-1, and the latter two genotypes were at par. At 10 gl⁻¹ agar, IWA-1 had taller plantlets, heavier foliage dry weight, root volume, as well as root dry weight than Konafubuki and Norin-1, whereas the latter two genotypes were at par for all these characteristics. This pattern was similar to the reported pattern of these genotypes for root dry weight under field conditions. However, such similarity in the in vitro and field behavior of the tested genotypes was not observed when nodal cuttings drawn from in vitro plantlets were used as explants. It is concluded that in vitro screening of potato under specific and limited water stress conditions by raising plantlets from nodal cuttings drawn from in vivo grown plants may provide a system for effectively differentiating the genotypes for their expected root mass production under field conditions.     

Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

A comparison of in vitro with in vivo flowering in Gentian

Young nodal explants of Gentiana triflora Pall. var. axillariflora were cultured in a woody plant medium (WPM) supplemented with B5 vitamins, sucrose (3%) and kinetin (2.0 μM). A novel observation was made in that in vitro flowering occurred following development of the axillary bud of the cultures. A comparison was made between in vitro and in vivo flowers. Although smaller in size, the in vitro flowers were morphologically comparable to the in vivo ones. Flowers from both sources were semi-opened or not opened. The colour of the in vitro flowers was paler than those in vivo. Stigma development was generally poor in both in vitro and in vivo flowers. Pollen viability was over 90% in both types of flowers. About 11% and 34% of pollen from in vitro and in vivo flowers, respectively, germinated on WPM containing 100 g l⁻¹ sucrose solidified with 10 g l⁻¹ agar. Hand pollination of stigma could raise viable seed production in in vivo-flowering plants from 0.3 o/o (i.e. without aided pollination) to 3% but none in in vitro-flowering plants where only seed-like structures, probably unfertilised ovules, were found. This revised version was published online in August 2006 with corrections to the Cover Date.     

High-efficiency phosphinothricin-based selection for alfalfa transformation

Despite the significant advantages of using herbicide resistance for selection of genetically engineered plants, alfalfa transformation has relied primarily on selection for antibiotic resistance. In the few studies reporting the use of resistance to the herbicide phosphinothricin (PPT), transformation efficiencies were low. The present investigation describes a PPT-based selection system for alfalfa transformation that uses the phosphinothricin acetyl-transferase (pat) gene as a selectable marker and 5.0 mg l⁻¹ of bialaphos as the selective agent. The method achieves transformation efficiencies, measured as the percentage of explants giving rise to one or more transformed plantlets, greater than 50%. These plantlets accumulated detectable amounts of PAT at levels varying from 2 to 1367 pg μg⁻¹ total protein. Transformed plants transferred to soil in the greenhouse were phenotypically normal and exhibited resistance to bialaphos leaf painting at 5 g l⁻¹ and applications of PPT equivalent to field-level use (0.5 kg ha⁻¹).     

Induction of Multiple Bud Clumps from Inflorescence Tips and Regeneration of Salt-tolerant Plantlets in Beta vulgaris

We developed an efficient method for sugar beet multiplication in vitro from excised immature inflorescence tips. On Murashige & Skoog medium supplemented with 4.4 μM 6-benzylamino- purine and 1.3 μM naphthaleneacetic acid, multiple bud clumps were induced from segments of inflorescence tips. The clumps proliferated rapidly. By radiation of small bud clumps at an appropriate dose and by directional selection for NaCl tolerance, we obtained salt-tolerant bud clumps and regenerated plantlets. The plants were vernalized and self-pollinated. The seeds of the regenerated plants were sown in pots of sand and irrigated every day with a solution of 342 mM NaCl. Some of the seeds germinated and grew normally in the 342 mM NaCl solution, exhibiting higher salt-tolerance than the control ones; such seedlings after the saline selection were transplanted to soil and the plants grew normally and produced plump root tuber similar to controls. The seeds from two selected lines germinated and grew for a few weeks in 513 mM NaCl solution before the seedlings withered. In saline soil where the salt concentration was about 154 mM, the yields of tuber from the plants of three salt-tolerant lines were about 45–50 tons ha⁻¹, approximately 2.6–2.9 times of the controls. It is concluded that we have got salt-tolerant materials with good agronomic traits for sugar beet breeding via selection in vitro.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.) from leaf explants and accumulation of esculin

Summary An efficient protocol has been developed for the regeneration of plantlets from leaf explants of witloof chicory (Cichorium intybus L.). Regeneration via callus was obtained on modified Murashige and Skoog semisolid medium (MS) containing 2.0 μM indole-3-acetic acid +5.0 μM 6-furfurylaminopurine (kinetin), and 1000 mgl⁻¹ casein hydrolyzate. At least five or more shoots regenerated from each callus. The shoots were rooted on MS +0.2 μM indole-3-butyric acid. The plantlets thus obtained were successfully established in soil after bardening. Esculin accumulation was recorded in plant tissues at different stages of differentiation in in vitro cultures and compared with in vivo-grown, plants. The esculin accumulation was higher in in vitro plants.     

Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Selection and breeding for salinity tolerancein vitro

Summary Selection for tolerance to NaCl inCitrus sinensis andC. aurantium has been carried out in agar and suspension cultures. Callus was subjected to culture media containing up to 0.17M NaCl for ten passages. Selected cell lines were grown for three passages on media without salt before further tests on saline media. Four stable tolerant cell lines, differing in degree of tolerance, have been selected fromC. sinensis. Four lines of similar tolerance have been selected fromC. aurantium. The stability of most lines was very satisfactory. MostC. sinensis lines grew well in media containing up to 0.2M NaCl, andC. aurantium lines in media of up to 0.15M NaCl.Embryos were regenerated in most selected cell lines fromC. sinensis and, more sporadically, fromC. aurantium. Addition of 0.5–0.6% NaCl to the media often enhanced embryogenesis. Embryos from a selected line ofC. sinensis showed higher tolerance to NaCl in the medium than comparable embryos from an unselected line.Single embryos derived from both selected and unselected cell lines ofC. sinensis were successfully cloned. A limited comparison of plantlets from one tolerant line (R14) with plantlets from unselected control lines showed better adaptation of the former to salt (0.085 to 0.12M NaCl in the medium), and a lesser degree of leaf burn symptoms.Contribution No. 1045-E, 1984 series.     

In vitro propagation of Notocactus magnificus

Most commercially grown cacti can be easily propagated by seed and/or cuttings. A group of rare and endangered species does not fit into this category and is therefore a good candidate for in vitro propagation productions as a tool to overcome habitat and plant-destruction. The number of rare and endangered species of Cacti goes into about 100. Many show a low production and germination of seeds and plantlets are prone to damping-off, making the in vitro propagation a feasible alternative for the multiplication and conservation of their germplasm. The aim of the present investigation is to establish a protocol for the in vitro culture and plant regeneration of Notocactus magnificus, the blue cactus, a highly ornamental species, native to Brazil. The surface sterilization of the explants was achieved with immersion for 10 min in sodium hypochlorite solution for either seeds (0.25% v/v) or ribs segments (1% v/v). Callus formation was observed when explants were cultured on MS medium supplemented with sucrose at 2% (w/v), 2,4-dichlorophenoxyacetic acid 0.5 μM, benzylaminopurine 4.4 μM, thiamine HCl 0.4 mg l⁻¹ and i-inositol 100 mg l⁻¹. The regeneration of shoots was carried out on MS medium supplemented with either different concentrations of benzylaminopurine and 1-naphthaleneacetic acid, or kinetin and indole-3-acetic acid. The highest number of shoots occurred when MS medium was supplemented with benzylaminopurine 22.2 μM, sucrose 3% (w/v) and agar 0,6% (w/v). In vitro spontaneous rooting of shoots was observed after eight months under culture on MS medium. Only in vitro rooted shoots developed into normal plants under glasshouse culture conditions. This in vitro protocol should be useful for the conservation as well as mass propagation of Notocactus magnificus.     

Changes in nitrogen content and protein profiles following <Emphasis Type="Italic">in vitro</Emphasis> selection of NaCl resistant mung bean and tomato

Mung bean and tomato were in vitro selected on media containing 0, 25, 50, 100 and 150 mM NaCl. Two types of media (hormone supplemented media, CB and hormone free media, MS) were used for mung bean using cotyledon explants whereas two types of explants (cotyledons and shoot apices) were used for tomato on MS media. Total-N, protein content, nitrite reductase (NiR) activity and protein protein profiles were checked in selected plants and compared to original non selected ones. NaCl at low concentrations slightly increased total-N in shoots and roots of in vitro selected mung bean and tomato whereas higher concentrations induced significant reductions. Similar increases in protein content were detected at lower concentrations with no significant effects thereover. On the contrary, NaCl gradually inhibited NiR activity. Similar responses of total-N, protein and NiR activity, but with greater magnitudes, were detected in original plants. In addition, NaCl significantly reduced dry weights of shoots and roots of either in vitro selected or, in particular, original intact plants. Moreover, electrophoresis (SDS-PAGE) of protein from shoots of either in vitro selected or intact plants showed that NaCl induced new protein bands while some others were concomitantly disappeared. The induction of one or more of the 86.4, 79, 77.6, 77 and 71.5 kDa bands following in vitro selection and/or the disappearance of the 86 kDa band from intact plants seemed necessary for mung bean resistance. Also, the presence of 86.2 kDa band and/or the loss of the 85.8 and 57.5 kDa bands might be included in tomato resistance. Of these induced bands in mung bean selected on CB media, only two bands were detected in plants selected on MS media. In tomato, two bands lost following selection from cotyledons but only one band lost following selection from shoot apices. These changes in protein pattern therefore might serve as adaptive regulators for resistance to NaCl.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Growth Performance of Cuttings Raised from in vitro and in vivo Propagated Stock Plants of Rosa damascena Mill.

Comparative studies on rooting and growth performance of cuttings raised from in vitro and in vivo grown plants of Rosa damascena are described. Cuttings were treated with different auxins and upon transfer to soil their growth performance was recorded. Overall, the auxin treated cuttings of in vitro raised plants responded better than the cuttings of in vivo raised plants. Optimal response for percentage of rooting, root number, root length and bottom breaks was observed at 100 mg dm^–3 IBA. The cuttings derived from in vitro raised plants showed a significantly better response for percent rooting, root number, root length and bottom buds in control treatments.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors affecting Agrobacterium tumefaciens-mediated genetic transformation of Lycium barbarum L.

Summary Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation, including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included preculture of leaf explants from 3-wk-old seedlings for 3 d on the medium for callus induction followed by inoculation with Agrobacterium strain EHA101 (pIG121 Hm), co-cultivation for 3d at 24&#x00B0;C, and transfer to the selection regeneration medium with 50 mg l^&#x2212;1 kanamycin (Kan). Using this protocol, 65% L. barbarum explants gave rise to Kan-resistant and GUS-positive calli. In addition, the expression of introduced transgene (npt II) in clonal progeny was verified by formation of calli and somatic embryos from leaf segments of nine transgenic plants grown on the Kan-containing medium. All explants formed calli at 50 mg l^&#x2212;1 Kan and seven out of nine transgenic plants were found to possess callus-forming capacity even at 100 mg l^&#x2212;1 Kan. These calli also possessed higher SE potential on SE medium supplemented with 25 mg l^&#x2212;1 Kan.     

Micropropagation of <Emphasis Type="Italic">eclipta alba</Emphasis> (L.) hassk—An important medicinal plant

Summary An efficient and reproducible protocol for mass propagation of Eclipta alba (L.) Hassk, an important medicinal plant, was standardized by culturing shoot tips and nodal segments taken from in vitro raised plants. Maximum shoot proliferation occurred when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ benzylaminopurine (BAP). The shoot buds formed were further multiplied and maintained on medium containing BAP (0.5 mgl⁻¹) and gibberellic acid (0.5 mgl⁻¹). Rooting was best achieved on MS medium supplement with 1 mg⁻¹ indole-3-butyric acid. Rooted plantlets attained maturity and flowered normally in the field.     

Mutation induced by ethylmethanesulphonate (EMS), in vitro screening for salt tolerance and plant regeneration of sweet potato (<Emphasis Type="Italic">Ipomoea</Emphasis><Emphasis Type="Italic">batatas</Emphasis> L.)

Salt tolerant cultivars of sweet potato (Ipomoea batatas L.) can be obtained from induced mutation. The objective of the present study was to induce mutation for salt tolerance using ethylmethanesulphonate (EMS) in calli of sweet potato, followed by cell line selection and subsequent plant regeneration. Calli initiated from leaf explants were treated with 0.5% EMS for 0, 1, 1.5, 2, 2.5 and 3 h, followed by rinsing with sterile distilled water for four times. Preliminary experiments showed that 200 mM NaCl could be used as selection pressure. Salt tolerant calli were sub-cultured on medium supplemented with 200 mM NaCl for selection of mutant cell lines and this process repeated 5 times (20 days each). The selected calli were transferred onto somatic embryo formation medium, which was Murashige and Skoog (MS) medium supplemented with 4 mg l⁻¹ abscisic acid (ABA), 10 mg l⁻¹ gibberellic acid (GA). After 15 days, somatic embryos were transferred onto MS medium supplemented with 0.05 mg l⁻¹ ABA, 0.2 mg l⁻¹ zeatin (ZT) for regeneration. Plants designated as ML1, ML2 and ML3 were regenerated from the somatic embryos formed by calli treated with 0.5% EMS for 2 and 2.5 h. After propagation, salt tolerance of these mutants was investigated. Data suggested the mutants were more salt tolerant than control plants.