Photoautotrophic growth of sugarcane plantlets <Emphasis Type="Italic">in vitro</Emphasis> as affected by photosynthetic photon flux and vessel air exchanges

Summary Explants of sugarcane, a C₄ plant, were cultured in vitro for 18d on Floridalite (a solid cube consisting of vermiculite and cellulose fibers) used as supporting material with sugar-free Murashige and Skoog liquid medium with double-strength KH₂PO₄, MgSO₄, FeSO₄, and Na₂-EDTA in the vessel with enhanced natural ventilation. CO₂ concentration in the culture room was kept at 1500 μmol mol⁻¹ (four times the atmospheric CO₂ concentration) during the photoperiod. A factorial experiment was designed with two levels of photosynthetic photon flux (PPF) and three levels of N (number of air exchanges of the vessel). The results were compared with those in the control treatment (photomixotrophic culture using sugar-containing agar medium under low PPF and low N). PPF and N showed significant positive effects on the growth of sugarcane plantlets in vitro. In the photoautotrophic (using sugar-free medium) treatments with relatively high PPF (200–400 μmol m⁻² s⁻¹) and high N (2–10 h⁻¹), the growth of plantlets was four to seven times greater than that in the control. Also, the culture period for multiplication and rooting was shortened from 30 d in the control to 18 d or less in the photoautotrophic, high PPF, and high N treatments. Use of porous supporting material in photoautotrophic treatments promoted rooting and plantlet growth significantly.     

Opposite effects of exogenous sucrose on growth, photosynthesis and carbon metabolism of in vitro plantlets of tomato (L. esculentum Mill.) grown under two levels of irradiances and CO2 concentration

The long-term effects of exogenous sucrose (3 percnt;) on growth, photosynthesis and carbon metabolism ofin vitro tomato plantlets were investigated under two sets of growth conditions that respectively favor source- or sink-limitations of photosynthesis: 1) low photosynthetic photon flux (PPF) (50 μmol m⁻² · s⁻¹) and low CO₂ concentration (400 μmol mol⁻¹) and 2) high PPF (500 μmol m⁻² · s⁻¹ and high CO₂ concentration (4000 μmol mol⁻¹). The supply of sucrose under source-limitation conditions increased the growth, the maximal photosynthetic rate, the chl content, the maximal quantum yield of Photosystem II estimated by the Fv/Fm chl fluorescence ratio as well as the soluble sugars (hexoses, sucrose) and starch contents in roots, young and mature leaves when compared to those of photo-autotrophic plantlets. Also, sucrose feeding under these conditions strongly increased the activity of sucrose synthase (SS) (EC 2.4.1.13) in roots and young leaves whereas the activities of sucrose phosphate synthase (SPS) (EC 2.4.1.14), acid invertase (INV) (EC 3.2.1.26) and ADP-glucose pyrophosphorylase (ADPGppase) (EC 2.7.7.27) were highly stimulated in roots and mature leaves. Contrary to these observations, the supply of sucrose to plantlets developed under high PPF and CO₂ concentration decreased growth and led to a somewhat lower maximal photosynthetic rate relative to photo-autotrophic plantlets. These negative responses to exogenous sucrose were accompanied by stronger accumulations of hexose and starch, larger stimulation of INV in mature leaves developed under conditions of sink limitation than those from source limitation conditions. Moreover, under high PPF and high CO₂ concentration, exogenous sucrose led to a marked repression of the SPS activity and caused much lower stimulations of ADPGppase in mature leaves than those observed at low PPF and low CO₂ concentration. We therefore conclude that under our experimental conditions, the interactive effects of exogenous sucrose and environmental conditions on growth and photosynthesis could be rationalized by the source-sink equilibrium of thein vitro tomato plantlets.     

Photosynthetic and carbohydrate status of easy-and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes during In vitro culture and Ex vitro acclimatization

Summary The photosynthetic and carbohydrate status of an easy-to-acclimatize (EK 16-3) and a difficult-to-acclimatize (EK 11-1) genotype of Uniola paniculata L. (sea oats), a native dune species of the southeastern US, were evaluated during in vitro culture and ex vitro acclimatization. Net photosynthetic rate was eight times greater for EK 16-3 than EK 11-1 plantlets after ex vitro transfer. In vitro-produced leaves were morphologically similar to ex vitro-produced leaves and exhibited similar photosynthetic competence. EK 11-1 plantlets exhibited greater transpiration rates at the time of ex vitro transfer than EK 16-3 plantlets. However, the small magnitude of this difference, although significant, indicated that control of water loss was probably not the main cause for poor acclimatization of EK 11-1 plantlets. Carbohydrate analysis in vitro revealed that EK 16-3 plantlets utilized leaf starch reserves more rapidly than EK 11-1 plantlets. Starch utilization correlated with the development of leaves with expanded leaf blades during in vitro rooting in EK 16-3 plantlets. After ex vitro transfer, both genotypes exhibited significant decreases of starch and soluble sugar content in shoots and roots. However, the higher photosynthetic ability of shoots in EK 16-3 resulted in greater accumulation of shoot soluble sugars than EK 11-1 after 2-wk ex vitro culture. After 6-wk in vitro rooting, there were significantly higher chlorophyll and soluble protein contents, ribulose 1,5-bisphosphate carboxylase (rubisco) and phosphoenolpyruvate carboxylase activities in EK 16-3 than EK 11-1 shoots. These differences also correlated with the development of anatomical and morphological leaf features in EK 16-3 similar to those of greenhouse-produced leaves.     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.     

Environmental control for the large-scale production of plants through in vitro techniques

Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to have high photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather than on sugar-containing medium, provided that the environmental factors, such as CO₂ concentration, light intensity and relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets in vitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitro plantlets. Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants have been developed. Advantages of sugar-free over conventional (heterotrophic or photomixotrophic) micropropagation systems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets in vitro have less physiological and morphological disorders, biological contamination in vitro is less, plantlets have a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be used because of less biological contamination. Hence, production costs could be reduced and plant quality could be improved significantly with photoautotrophic micropropagation. Methods for the measurement and control of in vitro environments and the beneficial effects of environmental control on photosynthetic growth, development, and morphogenesis in large-scale production of micropropagated plantlets are presented. This revised version was published online in June 2006 with corrections to the Cover Date.     

不同培养条件对勺叶茅膏菜试管苗矶松素含量的影响

为探讨培养条件对勺叶茅膏菜(Drosera spatulata)试管苗矶松素积累的影响,采用高效液相色谱(HPLC)法测定矶松素含量,对不同器官和不同培养条件下的勺叶茅膏菜试管苗矶松素含量变化进行研究。结果表明,勺叶茅膏菜试管苗根的矶松素含量显著高于叶片;光质和有机物含量对勺叶茅膏菜试管苗矶松素含量的影响不显著,但对试管苗的生长具有显著影响,最佳培养光质为白光,其次为红光和蓝光,最后为绿光;适当降低培养基中有机物含量可促进勺叶茅膏菜试管苗的生长发育;植物生长调节剂对矶松素积累的影响效应依次为6-BANAAKTGA3,而对试管苗生长的影响效应依次为6-BAGA3NAAKT。因此,勺叶茅膏菜试管苗的最佳培养条件为:以1/2MS为基本培养基,添加0~0.2 mg L–1 6-BA、0.2 mg L–1 NAA、0.5 mg L–1KT和0.1 mg L–1 GA3,于白光下培养。     

Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions

Suboptimal environmental conditions inside closed culture vessels can be detrimental to in vitro growth and survival of plantlets during the acclimatization process. In this study, the environmental factors that affected Doritaenopsis plantlet growth and the relationship between growth and sugar metabolism were investigated. Cultures were maintained under heterotrophic, photoautotrophic, or photomixotrophic conditions under different light intensities and CO₂ concentrations. Photoautotrophic growth of Doritaenopsis hybrid plantlets could be promoted significantly by increasing the light intensity and CO₂ concentration in the culture vessel. The concentration of different sugars in the leaves of in vitro-grown plantlets varied with different cultural treatments through a 10-wk culture period. Starch, reducing sugars, and nonreducing sugar contents were higher in plantlets grown under photoautotrophic and photomixotrophic conditions than in heterotrophically grown plantlets. Net photosynthesis rates were also higher in photoautotrophically and photomixotrophically grown plantlets. These results support the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high photosynthetic photon flux density. Growth was greatest in plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions with high photosynthetic photon flux density. The physiological status of in vitro-grown Crassulacean acid metabolism (CAM)-type Doritaenopsis showed a transition from C3 to CAM prior to acclimatization.     

Similarity of growth patterns between plantlets and seedlings of Brassica campestris L. under different in vitro environmental conditions

Explants and seeds of Brassica campestris L. were cultured on Murashige & Skoog (1962) medium with and without sucrose in a vessel with different numbers of air changes per hour under different PPF (photosynthetic photon flux) conditions. The growth and development of plantlets in the vessel were similar to those of seedlings when cultured under the same in vitro environmental conditions. The growth and development of seedlings when cultured under the same in vitro environmental conditions. The growth and development of plantlets/seedlings were greater for treatments with a higher number of air changes per hour and a higher PPF regardless of the sucrose concentration in the culture medium.The CO₂ concentration in the vessel with a lower number of air changes per hour decreased to approximately 100 ppm during the photoperiod on day 21 due to the photosynthetic activities of the plantlets/seedlings. The low CO₂ concentration, in turn, reduced the net photosynthetic rate of plantlets/seedlings in the vessel, and thus affected their growth and development.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration in the culture room - MS mineral composition of Murashige & Skoog (1962) medium - PPF photosynthetic photon flux     

Number of air exchanges,sucrose concentration,photosynthetic photon flux,and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro

Rehmannia glutinosa plantlets were cultured for 4 weeks under different culture conditions to determine the optimum environment for in vitro growth and ex vitro survival. Plantlet growth increased with an increasing number of air exchanges of the culture vessel, exhibiting greatest shoot weight, total fresh weight, leaf area, and chlorophyll content at 4.4 h⁻¹ of air exchanges. High sucrose concentration (30 g l⁻¹) increased root weight but reduced shoot growth. Net photosynthetic rates of the plantlets were greatest when sucrose was not added to the medium. On the other hand, ex vitro survival of the plantlets was not influenced by sucrose concentration. In the experiment on difference in photoperiod and dark period temperatures (DIF) and photosynthetic photon flux (PPF), plantlet growth increased as DIF and PPF levels increased. Particularly, increasing PPF level had a more distinctive effect on plantlet growth than increasing DIF level. The interaction of DIF × PPF was also significant, showing the greatest plantlet growth in positive DIF (+8 DIF) and a high PPF (210 μmol m⁻² s⁻¹). In conclusion, the results of this experiment suggest that increased number of air exchanges of the culture vessel, decreased sucrose concentration, and positive DIF in combination with high PPF level enhanced growth and acclimatization of Rehmannia glutinosa plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

Photosynthetic characteristics of Cymbidium plantlet in vitro

The photosynthetic characteristics of the Cymbidium plantlet in vitro cultured on Hyponex-agar medium with 2% sucrose were determined based on the measurements of CO₂ concentration inside and outside of the culture vessels. The CO₂ measurements were made with a gas chromatograph at a PPF (photosynthetic photon flux) of 35, 102 and 226 mol m^-2 s^-1, a chamber air temperature of 15, 25 and 35°C and a CO₂ concentration outside the vessel of approximately 350, 1100 and 3000 ppm. The net photosynthetic rates were determined on individual plantlets and were expressed on a dry weight basis. The steady-state CO₂ concentration during the photoperiod was lower inside the vessel than outside the vessel at any PPF greater than 35 mol m^-2s^-1 and at any chamber air temperature. The photosynthetic response curves relating the net photosynthetic rate, PPF, and CO₂ concentration in the vessel and chamber air temperature were similar to those for Cymbidium plants grown outside and other C₃ plants grown outside under shade. The results indicate that CO₂ enrichment for the plantlets in vitro at a relatively high PPF would promote photosynthesis and hence the growth of chlorophyllous shoots/plantlets in vitro and that the plantlets in vitro would make photoautotrophic growth under environmental conditions favorable for photosynthesis.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration outside the vessel (in the culture room) - PPF photosynthetic photon flux     

Effects of sucrose concentration,supporting material and number of air exchanges of the vessel on the growth of in vitro coffee plantlets

Growth of coffee (Coffea arabusta) plantlets cultured in vitroas affected by sugar, types of supporting material and number of air exchanges of the vessel was investigated. Single node cuttings of in vitro coffee plantlets were cultured on half strength MS medium with or without 20 g l⁻¹ sucrose. Two types of supporting material, agar and Florialite, and two levels of air exchange expressed by number of air exchanges per vessel, 0.2 and 2.3 h⁻¹, were studied. At the end of a 40-day culture period, fresh weight, shoot length, root length and leaf area of plantlets when cultured on Florialite soaked in sugar-free medium and under the higher number of air exchanges were greater than those in sugar containing medium. Callus was observed at the shoot base of plantlets grown on agar medium containing sucrose. Photosynthetic ability of coffee plantlets in vitro was also significantly increased when grown on sugar-free medium with the high number of air exchanges and Florialite as a supporting material. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photoautotrophic shoot and root development for triploid melon

The aim of this investigation was to establish environmental factors which promote growth and photosynthesis of melon (Cucumis melo L.) shoot buds, in vitro, and determine if photoautotrophic shoots had superior root forming ability in photoautotrophic environments. Buds from the triploid melon clone ‘(L-14×B)×L-14’ were observed for 21 days after transfer from a multiplication MS medium with 3% sucrose and 10 μM benzyladenine (BA) to a shoot development medium with 1 μM BA at three levels of sucrose in the medium (0, 1 and 3%), and light (50, 100 and 150 PPF) and CO₂ (500, 1000 and 1500 ppm) in the culture chamber. More shoot buds were observed with 3% sucrose in the medium. Increased light and CO₂ had a positive interaction with shoot proliferation. Fresh and dry weights were greatest at 3% sucrose, 150 PPF light and 1500 ppm CO₂. Shoot buds grew more slowly in sugar-free medium, but fresh and dry weight still doubled over 21 days of culture. Net photosynthetic rates (NPR) of buds were negative after four days in treatment conditions, but became positive after transfer to fresh, sugar-free medium. Two triploid genotypes of melon were (1) grown in vitro with sugar (photomixotrophic) and without sugar (photoautotrophic), (2) rooted in sugar-free media, both in a laboratory controlled environment chamber (in vitro) and a greenhouse acclimatization unit (ex vitro), and (3) compared for subsequent nursery growth in the greenhouse unit. The genotype ‘(L-14×B)×L-14’ produced more shoots than ‘(L-14×B)×Mainstream’ in both photomixotrophic or photoautotrophic conditions. ‘(L-14×B)×L-14’ rooted as well from either photoautotrophic and photomixotrophic shoots but ‘(L-14×B)×Mainstream’ rooted less frequently from photoautotrophic shoots. Seventy-six percent of the shoots in the laboratory controlled environment chamber were able to root photoautotrophically, whereas 47% of the shoots in the greenhouse acclimatization unit were rooted. Between 77% and 88% of plantlets from all treatment combinations survived transfer to the nursery. After growth in the nursery, the sizes of plants (fresh weight, dry weight, leaf area) were the same for either genotype, from either photoautotrophic or photomixotrophic shoots. Nursery plants that had been rooted in the laboratory controlled environment chamber were larger than those rooted in the acclimatization greenhouse chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid in vitro propagation of Holarrhena antidysenterica using seedling cotyledonary nodes

A rapid in vitro propagation of Holarrhena antidysenterica has been developed. Seedling cotyledonary nodes on Murashige and Skoog medium (MS) containing 2 mg dm⁻³ N⁶-benzyladenine (BA) produced highest number of multiple shoots. The shoot numbers were increased further upon subculture on MS medium supplemented with 0.5 mg dm⁻³ BA. By repeated subculture of derived shoots, a high multiplication rate was established. The excised shoots were rooted on MS basal medium without growth regulators. The in vitro formed shoots were also rooted ex vitro by dipping them in 2 mg dm⁻³ of indole-3-butyric acid (IBA) solution for 2 min before transferring them onto the hardening medium. Successful hardening and further establishment (survival 90 %) of micropropagated plants under natural conditions was observed.     

Photosynthetic characteristics of coffee (Coffea arabusta) plantlets in vitro in response to different CO2 concentrations and light intensities

The photosynthetic characteristics of coffee ( Coffea arabusta) plantlets cultured in vitro in response to different CO₂ concentrations inside the culture vessel and photosynthetic photon flux (PPF) were investigated preliminarily. The estimation of net photosynthetic rate (P_n) of coffee plantlets involved three methods: (1) estimating time courses of actual P_n in situ based on measuring CO₂ concentrations inside and outside the vessel during a 45-day period, (2) estimating P_n in situ at different CO₂ concentrations and PPFs using the above measuring approach for 10-day and 30-day old in vitro plantlets, and (3) estimating P_n of a single leaf at different CO₂ concentrations and PPFs by using a portable photosynthesis measurement system for 45-day old in vitro coffee plantlets. The results showed that coffee plantlets in vitro had relatively high photosynthetic ability and that the P_n increased with the increase in CO₂ concentration inside the vessel. The CO₂ saturation point of in vitro coffee plantlets was high (4500–5000 μmol mol^-1); on the other hand, the PPF saturation point was not so high as compared to some other species, though it increased with increasing CO₂ concentration inside the vessel. This revised version was published online in June 2006 with corrections to the Cover Date.     

华重楼离体胚培养及植株再生初探

为探明华重楼离体胚培养及植株再生的基本体系,该文以华重楼离体幼胚为试验材料,以MS培养基为基本培养基,研究不同光照、不同浓度梯度组合的植物生长调节剂对华重楼离体幼胚萌发、成苗的影响。结果表明:培养到60 d时,暗培养条件下华重楼离体幼胚的生长率和萌发率分别比光培养条件下高45.25%、19.17%,故暗培养比光培养更有利于华重楼离体幼胚生长发育。当GA₃浓度相同时,离体幼胚萌发所需时间随IAA浓度增加而延长。不同浓度的GA₃都可促进离体幼胚萌发,促进作用由强到弱依次为5 mg·L^-1 GA₃>1 mg·L^-1 GA₃> 10 mg·L^-1 GA₃。在黑暗条件下,优选得到最适华重楼离体幼胚生长发育配方为1/2 MS+30 g·L^-1 蔗糖+7 g·L^-1琼脂+0.5 g·L^-1 活性炭+5 mg·L^-1GA₃+1 mg·L^-1 IAA。此配方可诱导华重楼离体幼胚在2个月左右萌发,萌发率达50%需80 d左右。在华重楼成熟胚出苗培养时发现,高浓度的植物生长调节剂会抑制华重楼成熟胚生长发育,高浓度GA₃甚至会导致华重楼成熟胚死亡。优选得到最适华重楼成熟胚出苗配方为MS+30 g·L^-1 蔗糖+6.2 mg·L^-1 硼酸+7 g·L^-1 琼脂+0.7 g·L^-1 活性炭+0.5 mg·L^-1 2,4-D+1.5 mg·L^-1 IAA + 1.5 mg·L^-1 ZT+5mg·L^-1 GA₃,诱导成熟胚出苗需43 d左右,75 d左右可形成真叶。     

An in vitro testing of chemoresistance in leukaemic patients

The fact that leukaemic cells are primarily or secondarily resistant to cytostatics is a serious phenomenon, which leads to the failure of chemotherapy of malignant diseases in clinical practise. Some detoxification and transporting systems are responsible for the generation of chemoresistance on the cellular level and the decrease of effectiveness in treatment. In vitro testing of chemoresistance of leukaemic cells is presently an inseparable component of “tailoring” therapy in the developing field of predictive oncology. The aim of this work was to estimate profiles of drug resistance, based on the predictive in vitro test, and to help in choosing the most effective cytostatic. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoline (MTT) assay was used, based on the direct effect of cytostatics on the viability of leukaemic cells in vitro. The number of living leukaemic cells was evaluated by a computer program, where LC₅₀ (concentration of cytostatics lethal to 50% of leukaemic cells) was established from the achieved dose-relation curves. Seventy-one samples of leukaemic cells isolated from the patients’ peripheral blood or bone marrow were examined. All samples were tested to 3 cytostatics minimally. It was found by the in vitro assay, that resistance to dexamethasone, prednisolone, etoposide and vincristine is increased in patients with acute myeloid leukaemia disease, compared to the acute lymphoblastic leukaemia patients. In patients with a relapsed disease population, leukaemic cells are highly heterogeneous in the MTT assay. It was concluded that the MTT assay can be used to study drug interactions in vitro in leukaemia samples. The type of interaction was highly different between patients, and depended on drug concentrations.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

Effect of aluminum on the in vitro activity of acid phosphatases of four potato clones grown in three growth systems

The aim of this study was to assess the effect of aluminum on the in vitro activity of acid phosphatases (APases) of four potato clones, Macaca and Dakota Rose (Al-sensitive), and SMIC148-A and Solanum microdontum (Al-tolerant), grown in vitro, in hydroponics or in a greenhouse. The enzyme was assayed in vitro in the presence of 0, 1.85, 3.70, 5.55 and 7.40 mM Al. In plantlets grown in vitro, root APases were inhibited by Al in all clones, while shoot APases were inhibited by Al in S. microdontum and Dakota Rose and increased in Macaca at all Al concentrations. In plantlets grown in hydroponics, root APases increased in Macaca at 1.85 mM Al, whereas decreased at all Al levels in S. microdontum. In greenhouse plantlets, root APases decreased at 7.40 mM Al in S. microdontum and SMIC148-A, and at 3.70, 5.55 and 7.40 mM Al in Dakota Rose. Shoot APases decreased in Macaca and SMIC148-A. Conversely, in Dakota Rose, APases increased at 1.85 and 3.70 mM Al. These results show that the effect of Al toxicity on in vitro APase activity depends not only on Al availability but also on the plant organ, genetic background, and the growth conditions. Therefore, it suggests that acid phosphatases activity assessed in vitro might not be a good parameter to validate the screening for adaptation of potato clones to Al toxicity.     

Indirect regeneration of Withania somnifera and comparative analysis of withanolides in in vitro and greenhouse grown plants

The present study reports an efficient protocol for indirect shoot organogenesis and plantlets regeneration of Withania somnifera (L.) Dunal. Leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The highest callus induction rate (89.5 %) and shoot regeneration rate (92 %) were obtained when 2 mg dm⁻³ BAP was combined with 0.5 mg dm⁻³ IAA. Three major withanolides (withaferine A, 12-deoxywithastramonolide and withanolide A) were investigated in different plant organs from in vitro and greenhouse grown plants. Leaves contained higher contents of withanolides and phenolics than roots or stems, whereas roots contained the highest contents of flavonoids and polysacharides. In vitro grown plants contained greater contents of phenolics, flavonoids and polysaccharides while lower contents of withanolides than greenhouse grown plants.