B5 Salts Research Articles

Production of podophyllotoxin, kaempferol and quercetin in callogenesis of Dysosma versipellis — an endangered medicinal plant

• Dysosma versipellis is an endangered medicinal herb found in southwest regions of China. • Callus initiation was achieved in B5 salts with 2 mg/L 2,4-D, and 0.2 mg/L kinetin under complete darkness. • The maximum callus biomass proliferation was obtained on B5 basal salts supplemented with 2 mg/L 2,4-D. • Leaf callus showed maximum production of podophyllotoxin, kaempferol and quercetin than that of control. • UPLC analysis revealed that roots and rhizomes were the primary source of secondary compounds accumulation. Dysosma versipellis is an endangered medicinal herb found in southwest regions of China. It has long been used in Traditional Chinese Medicine for general remedies in China. This herb is commonly used as an alternative to Podophyllum species. The large-scale removal of D. versipellis rhizomes from natural habitat continues at higher rates than natural regeneration. This study reports the callus induction from D. versipellis explants, callus proliferation and production of tumor inhibitory compounds such as podophyllotoxin, kaempferol and quercetin. The results revealed that tender leaves of D. versipellis exhibited the highest callus induction efficiency of 86% than other explants tested. The best callus initiation was obtained in Gamborg's medium (B5) augmented with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 mg/L kinetin, 3% sucrose, 0.1% activated charcoal and 0.7% agar and incubated under complete darkness. The maximum callus biomass proliferation was obtained on B5 basal salts supplemented with 2 mg/L 2, 4-D. The UPLC analysis of leaf callus showed maximum production of podophyllotoxin, kaempferol and quercetin than that of control callus. Furthermore, analysis of whole plant parts for their secondary compounds found roots and rhizomes were the primary source of secondary compounds accumulation. Therefore, this bioactive-enriched callus biomass protocol may be used as a possible technique for sustainable production of podophyllotoxin, kaempferol and quercetin at commercial scale.

Somatic embryogenesis mediated micropropagation of polyembryonic cultivars of mango Vellaikolumban and Olour using nucellus tissue

Abstract Somatic embryogenesis mediated micropropagation from nucellus tissue was attempted in polyembryonic mango at the ICAR-Indian Institute of Horticultural Research, Bengaluru during 2016–2019. Studies were conducted to optimize the different stages of somatic embryogenesis namely induction, proliferation, conversion, maturation, germination of somatic embryos and ex vitro establishment of plantlets using nucellus tissue. For culture initiation, various developmental stages of fruits ranging from less than 20 days post-pollination to more than 60 days-post pollination of cultivars Vellaikolumban and Olour were utilized. Fruits of 30–40 days post-pollination in Vellaikolumban and 40–50 days post-pollination in Olour gave better percentage callusing with fewer days for callus initiation and callus formation. Embryonic calli developed on induction medium containing Rugini olive (RO) constituents, 6% sucrose, and 5 ppm each of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and Giberellic acid (GA3). Upon shifting to proliferation medium (RO with 6% sucrose, 5 ppm each of 2, 4-D and GA3, 400 mgL−1of glutamine and 20% v/v coconut water) pro-globular and globular embryos were formed within 4–5 weeks. Further development of somatic embryos through early heart, late heart and cotyledonary stages occurred in conversion medium (half strength B5 salts, full RO organics, 5 ppm of Indole-3-Acetic Acid (IAA), 20% coconut water and 200 mgL−1 of casein hydrolysate). Maturation of embryos was obtained by using 0.01 mg L−1of Abscisic acid (ABA) and 100 mg L−1of Polyethylene glycol (PEG). Germination of embryos with shoot and root initiation was observed in the presence of 5 ppm of zeatin. Our results show that somatic embryogenesis can be used as an alternative method to conventional propagation methods for rapid multiplication of uniform planting material in polyembryonic mango cultivars.

Enhanced Alkaloid Production from Cell Culture System of Catharanthus roseus in Combined Effect of Nutrient Salts, Sucrose and Plant Growth Regulators

Callus and biomass culture of Catharanthus roseus L. were established to check for the presence of total alkaloid and its subsequent yield. Various treatments like strength of nutrient salts, sucrose concentrations and combinations of plant growth regulators (PGR’s) were applied to both MS and B5 in agar as well as suspension medium to test their effects on enhanced alkaloid content and its yield. There was no significant difference in any of the observable parameters of fresh wt, dry wt, alkaloid content, production, productivity and yield if the culture were treated similarly in both types of media formulations (MS or B5 salts). Physical state (agar solidified or the liquid suspension) of the medium had significant effect on all the parameters particular on fresh wt, alkaloid content and yields. Although, the fresh wt. and dry wt. of biomass in suspension culture was 2-3 times less than that of callus obtained from agar medium. However, the alkaloid content and yield was 2-3 times higher in suspension culture compared to agar medium in similar treatments. The highest alkaloid content observed was 5.67mg/g dwt in B5 suspension medium containing 3% sucrose and modified with 0.5mg/l 2,4-Dichlorophenoxy acetic acid (2,4-D) + 1 mg/l Kinetin (KIN) + 2mg/l α- naphthalene acetic acid (NAA). The combined effects of these factors on the enhanced production of total alkaloids were expected to contain higher yield of anticancer vinblastine and vincristine in the cell suspension culture system.

Somatic embryogenesis induction of litchi (Litchi chinensis Sonn.) from leaves of mature trees

Induction and development somatic embryos of litchi (Litchi chinensis Sonn.) from leaflet explants obtained from mature litchi trees were investigated. Somatic embryogenesis was significantly influenced by the cultivar, plant growth regulators, position of leaflet and orientation of leaf explants. Adaxial-face up orientation of leaf explants significantly enhanced embryogenesis in comparison with adaxial-face down orientation. Maximum number of somatic embryos were obtained using median position from basal leaflet with adaxial-face up orientation cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 2, 4 dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) (37.67). Two types of salts media (MS and Gamborg’s B5) as well as different sucrose concentrations were examined for development and maturation of somatic embryos. MS salts medium with 45g/l sucrose was effective on size of mature somatic embryos (8.00 > 0.5 cm SE and 6.33 < 0.5 cm SE) compared with B5 salts medium (4.33 > 0.5 cm SE and 4.66 < 0.5 cm SE) on the same concentration of sucrose. Development of somatic embryos occurred on the media without growth regulators, heart and torpedo stage somatic embryos were obtained. Somatic embryos enlarged more and turned to milky opaque in color and finally reached to mature milky cotyledonary stage. Scanning electron microscopy revealed different morphological development of somatic embryos. In addition, Anatomical observation showed differences in the embryogenic and non-embryogenic cells. Furthermore, anatomical observation for development stages of somatic embryos.

Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress

Anther culture can be used as a powerful tool to produce doubled haploid (DH) lines in medicinal plants, thus accelerating breeding programs. In the particular case of borage (Borago officinalis L.), a method to produce DH plants has not been yet published. In this work we evaluated the effect of different culture media and of different chemical (colchicine and n-butanol) and physical stresses (centrifugation and electroporation) on androgenesis induction and plant regeneration in borage anther cultures. We found that the highest response can be obtained with culture medium containing B5 salts and NLN vitamins, the addition of 200 mg/l colchicine during 4 days, a pretreatment of anthers with 0.2% n-butanol for 5 h, or the application to anthers of single physical stresses (either centrifugation at 300×g or a 100 V electrical shock, but not combined). This is the first report on the production of DH plants in borage. Together, the results presented hereby can be used as a basic framework for large-scale generation of DH plants in this important medicinal species.

Agrobacterium-mediated transformation of thin cell layer explants of Scutellaria ocmulgee small: a rare plant with anti-tumor properties

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shoot-derived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6- benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 µM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the β glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

Regeneration of adventitious shoots from mature stored cotyledons of Japanese plum ( Prunus salicina Lind1)

Adventitious shoot regeneration from mature stored cotyledons of Japanese plum ( Prunus salicina Lind1) was achieved in vitro. The influences of the presence and absence of the light, different concentrations of thidiazuron (TDZ) and benzyladenine (BA) in the culture media, TDZ pretreatments and different basal salts on shoot regeneration were evaluated. TDZ was more effective in inducing shoot regeneration from mature stored cotyledons than BA. Dark incubation significantly increased the regeneration frequencies. Quoirin/Lepoivre (QL) basal salts stimulated shoot regeneration more than woody plant (WPM) or B5 salts did. The frequency of adventitious shoot formation varied among the varieties and the regeneration ability appeared to be genotype depended. The frequency of regeneration under the optimum tested conditions for ‘Bruce’, ‘Shiro’, ‘Redheart’, ‘Gladstone’ and ‘Early Golden’ cotyledons were 66.7%, 46.7%, 43.3%, 26.7% and 6.7%, respectively.

An Efficient Tissue Culture Regeneration System for Georgia Plume, Elliottia racemosa, a Threatened Georgia Endemic

Georgia plume, Elliottia racemosa Muhlenb. ex. Elliott, is an extremely rare small tree or shrub endemic to Georgia, which is being severely affected by habitat loss and lack of sexual recruitment. In vitro plant regeneration of Georgia plume has not been previously reported and may be a method for the conservation and propagation of this threatened species. Studies evaluated the effects of sterilization methods, explant types, medium composition, and light environment on plant regeneration. An efficient plant regeneration system was developed in which adventitious shoot buds were induced using young, expanding leaf explants placed on an induction medium supplemented with 10 μm thidiazuron and 5 μm indole-3-acetic acid with Gamborg's B5 salts. Shoot elongation was promoted on a medium with 25 μm (2-isopentenyl) adenine incorporated into Woody Plant Medium. In vitro rooting studies evaluated continuous and pulse auxin treatments and ex vitro rooting trials after KIBA (indole-3-butric acid, potassium salt) dips. A 5-day pulse treatment on 100 or 150 μm indole-3-butyric acid produced ≈90% rooting of shoots with greater shoot and root dry weight than other pulse times. High rooting frequencies were obtained under in vitro and ex vitro conditions with over 85% survival of plantlets transferred to greenhouse conditions. The culture protocol was found to be effective with material collected from mature specimens in the wild from divergent populations. Tissue culture appears to be a promising approach for the propagation and conservation of this rare and threatened plant.

In vitro regeneration and transient gene expression in mango cv. ‘Vellaikolumban’

SUMMARYA protocol to regenerate shoots through somatic embryogenesis was developed in the polyembryonic mango cv. ‘Vellaikolumban’, and the feasibility of gene transfer using the βglucuronidase (GUS) reporter gene was demonstrated. Young, 25–30 d-old fruits were found to be ideal for culture initiation. MS and B5 media were equally effective for initiation of nucellar cultures. A higher percentage of somatic embryo induction was obtained from nucellar cultures using B5 or MS medium supplemented with 20% (v/v) coconut water compared to MS medium without coconut water. Maturation of somatic embryos, 1.0 – 1.5 cm in length, was optimised in M3 medium composed of B5 salts, 400 mg l–1 L-glutamine and 1 mg l–1 abscisic acid. Faciation and necrosis of embryogenic cultures could be effectively controlled through the use of 0.1 mg l–1 salicylic acid. For germination of mature somatic embryos, with well-developed roots and shoots, a semi-solid medium was found to be superior to a bilayer medium. Various stages of nucellar culture were transformed with Agrobacterium tumefaciens strain LBA 4404 containing the binary vector, pCambia 2301, harbouring the npt II gene as a selectable marker and GUS as a reporter gene. During kanamycin sensitivity tests, it was found that 200 mg l–1 kanamycin completely inhibited regeneration of fresh new callus in nonco-cultivated embryogenic callus derived from nucellus. Among the various stages of nucellar culture tested, embryogenic callus was found to be amenable to Agrobacterium-mediated gene transfer. Maximum transformation was obtained using 150 µl bacterial culture with 3 d of co-cultivation. The expression of the reporter transgene was confirmed by GUS assay.

In vitro regeneration and genetic transformation of the berberine-producing plant, Thalictrum flavum ssp. glaucum.

Protocols have been developed for the in vitro regeneration and Agrobacterium-mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum. Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l-1 sucrose, 2 mg l-1 kinetin, and 3 g l-1 Gelrite) containing 25 mg l-1 hygromycin B as the selection agent and 250 mg l-1 timentin to facilitate the elimination of Agrobacterium. Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l-1 hygromycin B, 250 mg l-1 timentin, and 3 g l-1 Gelrite). Detection of the beta-glucuronidase (GUS) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl−1 (8.9μM) N6-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl−1, 0.54μM) and N6-benzyladenine (0.5mgl−1, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS1/3 semisolid medium supplemented with indolebutyric acid (1 mgl−1, 4.9 μM).

Effect of auxin, cytokinin, and sucrose on cell cycle gene expression in Arabidopsis thaliana cell suspension cultures

Cell suspension cultures of Arabidopsis thaliana (L.) Heynh. have been used to study the effect of auxin (α-naphthaleneacetic acid), cytokinin (kinetin), and sucrose on cell cycle gene expression. Auxin, cytokinin, and/or sucrose were re-added to culture media depleted for all three mitogens and the effects on the transcript levels of key cell cycle genes were monitored. Different cell cycle genes responded differentially to the mitogen supply. The cyclin-dependent kinase (CDK)-encoding gene CDKA;1 and the A- and D-type cyclins were induced by sucrose, whereas CDKB1;1 and CYCB1;1 were induced most strongly by the combined administration of auxin and cytokinin. The transcript levels of CDK inhibitors were up-regulated upon the addition of Gamborg's B5 salts to depleted medium. This induction was counteracted by the presence of auxin, cytokinin, or sucrose, depending on the CDK inhibitor. The obtained results are discussed in regard to the current knowledge on mitogen effects on cell cycle regulation in Arabidopsis.

Agrobacterium-mediated transformation of opium poppy, Papaver somniferum, via shoot organogenesis

Summary An efficient Agrobacterium -mediated protocol has been developed for the stable genetic transformation of intact opium poppy, Papaver somniferum L., plants via shoot organogenesis. Excised cotyledons were cocultivated with the disarmed A. tumefaciens strain GV3101 carrying the pBI121 binary vector, and incubated on an optimized shoot induction medium consisting of B5 salts and vitamins, 30 g L −1 sucrose, 2 mg L −1 6-benzylaminopurine, 5 mg L −1 AgNO 3 , and 3 g L −1 Gelrite. Except for the cocultivation medium, all formulations included 30 mg L −1 paromomycin as the selective agent, and 200 mg L −1 timentin to eliminate the Agrobacterium . Eight-week-old, paromomycin-resistant shoots were transferred to an optimized root induction medium consisting of B5 salts and vitamins, 0.5 mg L −1 indole-3-acetic acid, 0.5 mg L −1 indole-3-butyric acid, and either 5 mg L −1 AgNO 3 or 40 mg L −1 putrescine. About 15 % of the regenerated shoots developed roots within eight weeks. Regenerated plants were transferred to soil, where they grew normally and set seed. Detection of the neomycin phosphotransferase gene, the high levels of β-glucuronidase (GUS) mRNA and enzyme activity, and the cytohistochemical localization of GUS activity in all organs, confirmed the genetic transformation of the regenerated plants. The transformation process did not alter the normal alkaloid content of opium poppy; thus, the reported protocol could serve as a valuable tool to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis.

Somatic embryogenesis and plantlet regeneration from nucellar tissue of monoembryonic mango²

SummaryHigh frequency somatic embryogenesis from nucellar explants was achieved in the monoembryonic mango `Arka Anmol'. For culture initiation, immature fruits of 2–3.cm long about 30–40.d post-pollination were ideal. Culturing half ovules with intact nucellus gave better percentage callusing and more embryonic cultures than using excised nucellus. Among the basal media, Rugini olive (RO) medium followed by Gamborg's B5 (B5) was superior to the Murashige and Skoog (MS) medium for culture establishment and embryo induction. However, B5 medium was better than the others for embryo conversion and further development. Embryonic calli formed on establishment medium (RO, 6% sucrose, 2.g l–1 activated charcoal [AC], 2.g l–1 phytagel, 5 mg l–1 each of 2,4-D and GA3) on transfer at 2–3 weeks to expression medium (RO with 6% sucrose, 2 g l–1 AC, 2 g l–1 phytagel, 400 mg-1 glutamine [Gl], 1–3 mg l–1 2,4-D and 10 mg l–1 GA3) showed pro- and globular embryos in another 2–3 weeks. Further embryo development through heart and early cotyledonary stages occurred in a conversion medium (half strength B5 salts, full RO organics, 4% sucrose, 400 mg l–1 Gl, 2 g l–1 phytagel, 2 g l–1 AC, 100 mg l–1 casein hydrolysate, 20% [v/v] coconut water, 0.5 mg l–1 2,4-D and 10 mg l–1 GA3). Proliferating embryonic cultures could be maintained in this conversion medium giving a continuous supply of embryos. Embryo maturation was obtained in the presence of 1 mg l–1 abscisic acid. Germination with shoot and root development was obtained in presence of 0.01 to 0.1 mg l–1 thidiazuron. Partly senescent fruits about to drop or having just fallen could also be used as the source of embryonic cultures. Preliminary studies using other monoembryonic cultivars revealed genotype dependent differential responses.

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l-1 kinetin. The planting density was 2.5–3.0×105 protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures.

Growth and the Uptake of Sucrose and Mineral Ions by Transformed Root Cultures ofDatura stramonium, Datura Candida×aurea, Datura wrightii, Hyoscyamus muticusandAtropa belladonna

Growth and the uptake of sucrose, anions (Cl(-), NO3(-), PO4(3-), and SO4(2-)), and cations (K(+), Na(+), Mg(2+), Ca(2+), and NH4(+)) by transformed roots of Datura stramonium, Datura wrightii. Datura candida x Datura aurea hybrid, Hyoscyamus muticus, and Atropa belladonna were examined during batch culture over 28 days in modified 14 litre stirred tank reactors containing Gamborg's B5 salts medium. All cultures completely removed NH4(+) and PO4(3-) from the medium, and Mg(2+) was totally removed by the Datura cultures. The other ions were consumed at different rates depending on the culture but were never totally removed from the medium. Amounts for each ion consumed per gram increase in biomass are presented and compared between the different cultures. More NO3(-) than any other ion was consumed by each of the cultures. Concomitant with the uptake of sucrose was a slow release of glucose into the medium. Roots were found to contain only low levels of free sugars. Total hyoscyamine production ranged from 115 mg to 633 mg per reactor.

The effect of varying levels of Gamborg's B5 salts and temperature on the accumulation of starch and hyoscyamine in batch cultures of transformed roots of Datura stramonium

The partitioning of carbon between reserve polysaccharide and alkaloid secondary products was investigated in batch cultures of transformed roots of Datura stramonium grown in media in which the carbon substrate concentration was held constant and the level of mineral nutrients was varied. The growth and accumulation of starch and hyoscyamine was examined in roots grown at temperatures of 20°C, 25°C or 30°C in media containing 5% sucrose and levels of mineral nutrients varying from 1/4 to twice the standard level of Gamborg's B5 salts. The dry matter content was highest (up to 15% w/w) in roots grown at either 20°C or 25°C in medium of the lowest ionic strenth (1/4 B5 salts) and decreased as the ionic strength was raised (down to 7% w/w with 2 B5 salts). Up to half of this decrease could be accounted for by loss of starch from the roots. At 20°C and 25°C, the starch content of the roots grown in medium of the lowest ionic strength (1/4 B5) was 40 mg g-1 and 22 mg g-1 fresh weight respectively but decreased to less than 1 mg g-1 weight at either temperature when the ionic strength of the medium was raised to 2 B5. At 30°C, starch accumulation was severely inhibited in all media. In contrast, varying either the temperature or the ionic strength of the medium had only a small effect on hyoscyamine accumulation which remained at between 0.4–0.6 mg g-1 fresh weight. Although increases in the level of mineral salts had little effect on the hyoscyamine content of the roots, total yields however, increased due to stimulation of growth. Time course experiments showed that cultures grown at either 20°C or 25°C continued to accumulate both starch and hyoscyamine into late stationary phase.

886 PB 460 SOMATIC EMBRYOGENESIS FROM IMMATURE COTYLEDONS OF TEXAS BLUEBONNET

This study was initiated to test the embryogenic potential of immature cotyledons (3-5 mm long) of Texas bluebonnet (Lupinus texensis). The embryo initiation medium consisted of B5 salts and vitamins with 3% sucrose and 22.6 μM 2,4-D alone or in combination with 1-15 μM of various cytokinins. Within 15-20 days, globular embryos were formed on the distal end of the cotyledons. Eventually the entire cotyledon surface was covered by embryo-like structures. Addition of cytokinins to the medium did not increase the percentage of cultures which formed embryos. In fact, addition of thidiazuron severely inhibited embryogenesis. Following transfer to an embryo maturation medium (MS medium with 0.38 μM abscisic acid alone or in combination with benzyladenine or zeatin) for 10-14 days, the embryos were placed in MS medium supplemented with GA (2.9 μM) or glutamine (200 mg/liter) with or without activated charcoal (0.5%) for embryo germination and plantlet development. Most of the embryos exhibited precocious germination and well-developed roots but failed to produce normal shoots. Therefore, additional work is needed to improve embryo conversion frequency.

Factors Affecting the Growth and Hyoscyamine Production during Batch Culture of Transformed Roots ofDatura stramonium

The growth and hyoscyamine production of transformed roots of DATURA STRAMONIUM (cell line DS 1) were investigated in batch culture experiments at three different temperatures and in media of varying total ion composition and sucrose level. Growth rate was not greatly affected by the level of Gamborg's B5 salts or the sucrose concentration. Growth rates of roots in a range of media were similar at 25 degrees C (doubling time T (d) 1.2-1.6 days) and at 30 degrees C (T (d) 1.2-1.7 days) but roots grew slower at 20 degrees C (T (d) 1.9-3.5 days). The total root yield on a fresh weight basis was higher in full strength B5 media compared with half strength B5 media at all sucrose levels tested. However, the dry weight content of the roots increased significantly (4-20%) as the sucrose level in the medium was raised (2-10%) at each of the three temperatures. At 20 degrees C, the hyoscyamine content of the roots was higher than that at 25 degrees C or 30 degrees C at all sucrose levels tested and was unaffected by increasing sucrose levels. However, increasing the level of sucrose (1-5%) in either half or full strength B5 media at 25 degrees C or 30 degrees C increased the hyoscyamine content of the roots by up to eight fold. At 20 degrees C, the total yield of hyoscyamine per flask or per unit of sucrose supplied was also higher than at 25 degrees C or 30 degrees C with sucrose levels up to 5%. The highest rates of hyoscyamine production by cell line DS 1 were obtained with full strength B5 medium with 5% sucrose at either 20 degrees C or 25 degrees C. Under these conditions, rates of hyoscyamine formation of up to 7.4 mg/l/d were observed under batch culture conditions. The amount of hyoscyamine released into the culture medium was much greater at 30 degrees C (up to 14% of the total) than at either 20 degrees C or 25 degrees C (up to 4% of the total).

In Vitro Culture Responses of Alyceclover Genotypes on Four Media Systems

Tissue culture protocols for regeneration are widely variable among plant species. Without known techniques for in vitro regeneration of a given species, many molecular methods cannot be applied in crop improvement programs. In this study, the in vitro response of six genotypes of alyceclover, Alysicarpus vaginalis (L.) DC., was evaluated on two organogenic systems, B5 and MS basal salts plus growth regulators, and two embryogenic systems, L2 and SH basal salts plus growth regulators. Callus growth of genotypes on the L2‐based medium was significantly greater than on the other treatments, with significantly less growth on the MS callus induction medium. Shoot bud induction, but not embryo induction, was observed for all six genotypes on one or more media treatments. Genotype FL‐1 initiated shoot buds on three of the four media treatments, B5, L2, and MS. Genotype FL‐10 produced the highest percent of call! with induced shoot buds, with 63 and 89% for the B5 and MS treatments, respectively. Plants of four genotypes were regenerated after transfer to root induction or embryo germination medium. Genotype FL‐10 produced 128 and 19 plants when cultured on the L2 and B5 systems, respectively. Genotypes FL‐1, FL‐4, and FL‐100 produced a total of 24 plants when cultured on the MS system. All regenerated plants were phcnotypically normal in appearance.