Callus induction from leaves of different paulownia species and its plantlet regeneration

The experiment was carried out on five different species of Paulownia for callus induction from leaves. MS medium was adopted as basic medium, and from different combinations of NAA and BA the suitable media were determined for callus induction, bud differentiation, and root differentiation of five different species. MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5NAA+4BA, MS+0.3NAA+6BA, and MS+0.3NAA+8BA were suitable media of callus inductions of leaves, respectively, for Paulownia tomentosa, Paulownia australis, Paulownia fortunei, Paulownia elongata and P. tmentosa x P. fortunei, and MS+0.3NAA+12BA, MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA, and MS+0.7NAA+12BA were suitable media for bud differentiation from leaf callus respectively for above five species. The rooting media was determined as 2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, 1/2MS+0.3NAA, and 1/2MS+0.5NAA. These results provide reference data for breeding new fine va-rieties with different kinds of Paulownia protoplasts fusions.

黄金梨叶片再生体系建立研究

从外植体接种方式选择、暗培养时间、植物生长调节剂浓度和配比等方面研究了黄金梨叶片再生植株的影响因素。结果表明:适于黄金梨叶片再生的诱导培养基为MS+1.0 mg/L 6-BA+0.2 mg/L NAA,在此培养基上,叶面朝上接种的叶片经过15 d的暗培养转入光照培养,可获得45%的再生率和平均2.2个不定芽;1 cm以上的不定芽经1/2 MS+3.5 mg/L IBA诱导10 d后,转入1/4 MS无激素培养基继续培养可获得高达91.6%的生根率和5个以上的不定根数。

Effect of Polysaccharides Including Elicitors on Organogenesis in Protocorm-like Body （PLB） of Cymbidium insigne in vitro

Among orchids, Cymbidiums have got a very high demand in both cut flower and pot plant trade. In the present study the effect of some polysaccharides such as chitosan and NAG （n-acetyl-glucosamine） on organogenesis in protocorm-like-bodies （PLBs） of C. insigne was studied. Synthetic phytohormones such as BA and TDZ （cytokinins） and NAA （auxin） were used for comparison. PLBs of C. insigne were explanted on modified Murashige and Skoog medium supplemented with the single addition of chitosan and NAG, and the combination of BA （benzyladenine） and NAA （1-naphthaleneacetic acid） also with the combination of NAA and TDZ （thidiazuron） among different concentrations. Combination treatments of auxin and cytokinins, the highest percentage of PLBs formation was 73% and shoot formation was 67% when cultured on the medium supplemented with 1.0 mg L1 BA without NAA. Combination treatment of NAA and TDZ, the PLBs formation was 90% and shoot formation was 60% obtained from medium supplemented with 1.0 mg Lt of NAA ＋ 0.1 mg L^-1 TDZ. Single addition of chitosan and NAG with modified MS medium was more effective for new PLBs and shoot formation. The highest percentage of PLBs formation was 87% and shoot formation was 80% obtained from the medium supplemented with 0.1 mg L^-1 chitosan. On the other hand, the PLBs formation rate reached 93% and shoot formation rate was 87% obtained from the medium supplemented with 0.01 mg L^-1 NAG. Application of polysaccharides to in vitro orchid PLBs allows developing new PLBs and shoot to form plantlet without synthetic phytohormones.

Conservation of Iris bismarckiana Regel （Iridaceae） Using Plant Regeneration via Somatic Embryogenesis

In an attempt to propagate and conserve the rare, showy bulbous plants of Iris bismarckiana, newly recorded to the flora of Jordan and to contribute to the conservation the wild lris species in Jordan, a simple rapid, time consuming protocol has been developed using plant regeneration via somatic embryogenesis. Somatic embryogenesis was induced in zygotic embryo culture on Murashige and Skoog （MS） medium supplemented with 2,4-dichlorophenoxy acetic acid （8 mg/L） as the sole plant growth regulator, where both embryogenesis calli and somatic embryos were induced. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos successfully from embryo clusters and transferred onto fresh MS medium. Data obtained were analyzed as a complete random design with three replications. Calli fragments that were transferred to embryogenesis induction medium （EIM） produced white embryo-like globular structures within two weeks. Within three more weeks, clusters of structures at various stages of development could be found on the same callus. The applied technique is rewarding and encouraging for further research on the endangered wild species of Iris in Jordan.

Effect of Different Treatment on Anther Callus Formation and Browning in Balsam Pear （Mornordica charantia L.）

Brown callus derived from anther limited the application of anther culture in balsam pear. In order to establish a more perfect regeneration system from anther cultuer, the effects of low temperature pretreatment, 2,4-dichlorophenoxyacetic acid （2,4-D）, vitamin C （Vc） and silver nitrate （AgNO3） on callus induction and browning in anther culture of balsam pear （Momordiea charantia L.） were investigated. The results showed that after pretreatment at 4 ℃ for 1 day, callus induction rate was the highest and browning rate was the lowest. Anthers on MS medium supplemented with 2,4-D 0.5 mg/L formed more and better callus. The medium supplemented with Vc or AgNO3 was advantageous to the induction of callus and reduction of browning. When cultured on medium supplemented with 50 mg/L Vc or 5 mg/L AgNO3, callus induction rate was the highest and browning rate was rather low.

Effect of Different Hormone Combinations on Somatic Embryogenesis in Cotton Cultivar Xinluzao 33(Gossypium hirsutum L.)

[Objective] This study was conducted to evaluate the efficiency of somatic embryogenesis and plant regeneration for an upland cotton cultivar Xinluzao33 under the induction of different hormone combinations and thus to determine the optimal hormone combination. [Method] Calli of Xinluzao33(Gossypium hirsutum L.) were induced from seedling hypocotyl tissue by a range of DK and BK combinations. Embryogenic calli and embryos were induced on callus-inducing medium(CIM) without any hormones. Callus appearance and quality were compared to determine which medium was the optimal for callus induction. Embryogenesis ratio was calculated to determine which medium was the best for somatic embryogenesis and plant regeneration. [Result] Callus induction rate was 100% in all the 12 hormone combinations.The calli were yellow or kelly, and their texture was loose or soft under low concentrations of DK combinations, green or white, variably compact under high concentrations of DK combinations. The calli induced by BK combinations were kelly or green, covering creamy white substance. The best medium for callus induction was DK6(0.05 mg/L 2, 4-D and 0.10 mg/L KT). Embryogenic calli were successfully induced from all the combinations. The efficiency of embryogenic callus induction,embryogenesis, and plantlet regeneration were significantly different among the 12 combinations. The result showed that the embryogenesis ratio was the highest in BK3 combination(0.50 mg/L IBA and 0.50 mg/L KT), 72.86% of embryogenic calli differentiated into somatic embryos after being cultured on CIM for 80 d, and80.93% of the somatic embryos finally regenerated into plants on SEM(somatic embryo induction medium). [Conclusion] These results indicate that hormone combination BK3(0.50 mg/LIBA and 0.50 mg/L KT) was the best medium for somatic embryogenesis and plant regeneration from Xinluzao33.

Callus and Adventitious Buds Formation from Stem Explants of Balsam Pear （Momordica charantia L.）

The effects of sterilization pretreatment, sampling time and growth regulators on callus formation, explant contamination and adventitious bud induction from stems of balsam pear （Momordica charantia L.） were investigated. Sterilized for 6 rain with 0.1% （w/v） mercuric chloride solution is an essential sterilization method for stems. June and July proved to be better for material taking, followed by May. The callus formation rate of balsam pears reached the highest when medium added with IBA 1.0mg/L and BA 2.0 mg/L, while the optimum medium for adventitious bud induction was MS added with IBA 0.5mg/L and BA 4.0 mg/L. And it will provide theoretical and technical basis for rapid propagation and breeding.

Preliminary Investigation on Somatic Embryogenesis from Immature Cotyledon Explants of Shea （Vitellaria paradoxa G,）

Long juvenile phase and lack of effective protocols for large scale vegetative propagation are limitations to domestication and improvement of the shea tree. The present study seeks to develop a protocol for plant regeneration of shea （Vitellaria paradoxa） from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium （MS） containing 3% sucrose, 0.24% Phytagel, and various concentrations of 2,4-Dichlorophenoxyacetic acid （2,4-D） after four weeks of culture in darkness. Rates of embryogenic callus induction were significantly affected by the addition of 2, 4-D to the medium. Within 28 days of culture, the highest percentage of embyogenic calli （77.61%） occurred on MS media containing 0.45 ~tM of 2,4-D in the dark. Somatic embryos were obtained by culturing embryogenic callus （in the dark） on MS medium fortified with 3% sucrose, 0.24% phytagel and devoid of growth regulators. Culturing at 16 h photoperiod restricted both the induction of embryogenic calli cultures and somatic embryos. Somatic embryos germinated, developed shoots and rooted vigorously on MS medium devoid of growth regulators. Germinated plantlets were acclimatized, successfully.

Effect of Growing Conditions of Rice Donor Plants on Anther Culture in Vitro

An experiment was conducted to find the effect of three types of donor plants growing conditions （growth chamber, open space ＋ growth chambers and open space） on callus induction and subsequent plant regeneration and productive shoot regeneration from the anthers culture in vitro of four rice hybrid （1-2, 2-1, 7-1, 13-3） developed by Primorsky Scientific Research Institute of Agriculture. Five variants of N6 medium （N6-I, N6-2, N6-3, Mix-l, N6-4） were used as basal medium. Mean value of callus induction frequency on three types of conditions ranged from 5.68% to 9.44% and the difference was non-significantly. In general, callus derived from donor plants grown on condition of open space ＋ growth chambers showed significantly better performances for plant regeneration （0.23 green regenerants on anther and 3.77 green regenerants on callus） and productive shoot regeneration （0.06 productive regenerants on anther and 0.56 productive regenerants on callus）. Favourable conditions for donor plant growth in open space positively affect on callus induction and regenaration. It is possible to get assured results on many hybrids, but not the highest. In growth chamber, frequency of callus induction can be the maximal only on some samples, few hybrids are resulted in deficiency of callus induction.

Influence of Abiotic Elicitors on Accumulation of Thymol in Callus Cultures of Origanum vulgare L.

Callus cultures of Origanum vulgare L. were established from leaf discus on Murashige and Skoog （MS） medium containing different levels of growth regulators, i.e., 2,4-Dichlorophenoxyacetic acid （2,4-D）, Naphthalene acetic acid （NAA）, Benzyl Adenine （BA） and Kinetin （Kn） and incubated under dark condition. Callus tissues were employed to study the influence of abiotic elicitors on the production of thymol. Constant weights of callus （300 mg） were cultured on accumulation medium treated separately with each one of elicitors used （50 g/L sucrose, 200 mg/L NaC1 and 50 or 100 mg/L proline）. The fresh and dry weights of callus were recorded after six weeks. The result indicated that maximum production of fresh and dry callus weight were 1,014 mg and 46.20 mg respectively achieved at 0.5 mg/L 2,4-D and 3 mg/L BA adding to the medium. Dry callus tissues were extracted with 70% methanol and analyzed by HPLC to determine the concentrations of thymol. The addition of abiotic elicitors to MS medium caused significant reduction in fresh weight of callus compared with control treatment. The concentration of thymol in the callus cultured on control treatment was 146.6 ppm. The data showed that 50 or 100 mg/L proline produced the highest yield of thymol 181.48 ppm and 174.58 ppm respectively, followed by sucrose 162.9 ppm, whereas the treatment with NaCI caused reduction in thymol concentration to percentage of 50.56% compared with the control.

Evaluation of Three Sunflower （Helianthus annuus L.） Hybrids for Salt Tolerance in Vitro

This study aimed to induce callus from three sunflower （Helianthus annulus L.） hybrids, namely Anna, Alhaja and Kuds, and to evaluate their callus for salt stress tolerance. Cotyledons and hypocotyl were taken from seedling of these hybrids and cultured on MS media contained 2,4-D （0,0, 0.5, 1.0, 1.5 and 2.0 mg/L） and kinetin （0.0, 0.5, and 1.0 mg/L）. The cultures incubated at 25 ＋ I^C under light condition （1,000 Lux） for 16 h/day. After 6 weeks observations were taken on the response of cotyledons and hypocotyl to callus induction. The induced callus were cultured on the same MS media that contained appropriate concentrations of 2,4-D and kinetin for callus induction as well as contained various concentration of sodium chloride NaCI （0.0, 0.05, 0.1, 0.15 and 0.2%）. After six weeks callus fresh and dry weights, proline and total carbohydrates concentrations were measured. The results showed significant differences among the hybrids, explants, 2,4-D and kinetin concentration and significant interaction between them in their percentage response for callus induction. The results also revealed that fresh and dry weights were significantly reduced with increased NaCI concentration in the medium, hybrids showed significant differences in their response to salt stress. Proline and total carbohydrate concentration increased in callus as NaCI increased in the media. Significant interaction was showed between hybrids and NaCI concentration in these parameters.

Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design

The production of thermostable laccases from a native strain of the white-rot fungus Pycnoporus sanguineus isolated in Mexico was enhanced by testing different media and a combination of inducers including copper sulfate（CuSO4）.The best conditions obtained from screening experiments in shaken flasks using tomato juice,CuSO4,and soybean oil were integrated in an experimental design.Enhanced levels of tomato juice as the medium,CuSO4and soybean oil as inducers（36.8%（v/v）,3 mmol/L,and 1%（v/v）,respectively） were determined for 10 L stirred tank bioreactor runs.This combination resulted in laccase titer of 143000 IU/L（2,2＇-azino-bis（3-ethylbenzthiazoline-6- sulfonic acid）,pH 3.0）,which represents the highest activity so far reported for P.sanguineus in a 10-L fermentor.Other interesting media resulting from the screening included glucose-bactopeptone which increased laccase activity up to 20000 IU/L,whereas the inducers Acid Blue 62 and Reactive Blue 19 enhanced enzyme production in this medium 10 times.Based on a partial characterization,the laccases of this strain are especially promising in terms of thermostability（half-life of 6.1 h at 60 °C） and activity titers.