Synseed conception for short-term storage,germplasm exchange and potentialities of regeneration genetically stable plantlets of desert date tree (Balanites aegyptiaca Del.)

Synthetic seed has become a proficient tool that facilitates conservation as well as mass propagation of elite plant species by encapsulating somatic embryo or meristem tissue. Inclusion of meristematic tissues instead of somatic embryos hastens widespread utilization of this technology in recent years. Synseeds offer short term conservation of germplasms, provide readily available tissue source for easy mass propagation where each synseeds can virtually act as zygotic seeds giving rise to plantlets. This is the first report on synseed production following the confirmation of the genetic homogeneity in regenerated plantlets of Balanites aegyptiaca Del. (L.) using ISSR marker system. In this study, nodal segments, excised from in vitro proliferated shoot cultures developed from mother plant, were encapsulated in calcium alginate beads and the finest gel complexation was achieved using 3 % sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (80 %) for conversion of encapsulated nodal segments into plantlets was obtained on Murashige and Skoog (MS) medium containing 12.5 μM benzyladenine and 1.0 μM α-naphthalene acetic acid. Encapsulated nodal segments could be stored at low temperature (4 °C) up to 4 weeks with a survival frequency of 82 %. The regenerated shoots were rooted on half strength MS medium augmented with 1.0 μM indole-3-butyric acid. Well-developed plantlets regenerated from encapsulated nodal segments were acclimatized successfully with 90 % survival frequency. Fingerprinting profiles of the regenerated plantlets derived from synseeds and the donor plant were generated using a total of 20 ISSR primers, of which 14 primers produced distinct, reproducible amplified products. A total of 158 scorable bands were obtained from the complete amalgamation of primers and plantlets and 98.7 % bands were monomorphic across the plantlets which indicate that this micropropagated line derived from synseed is genetically stable and demonstrates the reliability of our protocol for short term conservation, germplasm exchange and distribution of identical plants.     

Micropropagation of <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> × <Emphasis Type="Italic">E. urophylla</Emphasis> AEC 224 clone

Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis  ×  E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 µM BA and 0.5 µM NAA for another 30 days. In these media, the explant oxidation rate was high (95 %). Thus, in order to reduce oxidation, different culture media were compared: WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-D were tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25 µM TDZ and 0.1 µM NAA during 30 days for callus induction and then with 5.0 µM BA and 0.5 µM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA (2.46, 4.90 and 7.35 µM). The highest rooting percentage (35 %) was obtained on medium containing 2.46 µM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %.     

Stimulation of in vitro organogenesis from epicotyl explants and successive micropropagation round in Cassia angustifolia Vahl.: an important source of sennosides

This report describes a protocol for the in vitro shoot induction and plant regeneration from epicotyl explants of Cassia angustifolia on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), Kinetin and 2-iP (0.5–10.0 μM). MS medium supplemented with BA (5.0 μM) was the most effective in inducing adventitious shoots and growth. The highest rate of shoot multiplication was achieved on MS medium supplemented with BA (5.0 μM) and Indole-3-acetic acid (IAA, 1.0 μM). The nodal segments excised from the shoots regenerated from BA (5.0 μM) and IAA (1.0 μM) were used as explants for next three round of micropropagation. The number of shoots significantly increased at successive round of micropropagation. For rooting, MS medium supplemented with 2.0 μM indole-3-butyric acid proved to be better than that supplemented with IAA or α-naphthalene acetic acid. The in vitro raised plantlets with well developed shoot and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse. About 52 plants (85 %) survived out of 60 plants transferred in garden soil.     

Micropropagation of Vitex negundo L. and Random Amplified Polymorphic DNA Analysis of Micropropagated Plants

A micropropagation protocol was established for a medicinal plant Vitex negundo. Genetic stability of micropropagated plants was investigated. Multiple shoots were induced from nodal explants cultured on Murashige and Skoog (MS) medium with 0.53 μM naphthalene acetic acid (NAA) and 11.0 μM benzyl aminopurine (BAP) along with additives (ascorbic acid, 283.9 μM; citric acid, 130.1 μM; and arginine, 143.6 μM). Shoots were further multiplied by repeated transfer of the mother explant. The shoots were further multiplied on MS medium + 0.57 μM indole-3-acetic acid (IAA) and 6.6 μM BAP. The micropropagated shoots were pulse treated with 122.5 μM indole-3-butyric acid (IBA), in liquid MS medium and then transferred to autoclaved soilrite. These rooted ex vitro. Shoots were also rooted in vitro on a half-strength MS medium + 2.45 μM IBA. The survival rate of in vitro rooted plantlets was poor during hardening compared to ex vitro rooted plantlets. About 95% of the ex vitro rooted, hardened plantlets survived in the field. Genetic stability of micropropagated plants was tested by using 25 random amplified polymorphic DNA primers. The cloned plants exhibited no variation in banding pattern in comparison with the mother plant.     

In vitro regeneration and the antioxidant enzymatic system on acclimatization of micropropagated Vitex trifolia L.

The objective of the study was to develop an in vitro shoot regeneration protocol by utilising shoot tips explant from Vitex trifolia L. Shoot tip explants obtained from a 3-year old plant was cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5 or 10.0 µM) of thidiazuron (TDZ). The optimal level of TDZ supplementation to the culture medium was 5.0 μM for 15 d induction period. The highest number of shoots (22.2 ± 0.1) and shoot length (5.1 ± 0.1 cm) were achieved when TDZ-exposed explants were sub-cultured on MS medium containing 6-benzyladenine (1.0 µM) and 0.5 µM α-naphthalene acetic acid (NAA) after 8 weeks of culture. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 µM NAA. During the acclimatization period, changes in activities of antioxidant enzymes were observed. Superoxide dismutase activity increased reaching maximum at 28th day after transplantation. Likewise, an upregulation of the catalase, ascorbate peroxidase and glutathione reductase enzyme activities were also observed. These observed changes reflected the ability of plants in developing an antioxidant enzymatic defence system aiding in survival against oxidative stress and in reducing release of free radicals. Plantlets were successfully hardened off and acclimatized in earthen pots containing garden soil with a survival rate of 90 %.     

An efficient in vitro process for recurrent production of cloned plants of Vitex negundo L

An efficient method for cultivation of Vitex negundo L. through axillary shoot (collected from micropropagated plants) proliferation has been successfully developed, which can be employed at a commercial scale. Axillary shoot induction was most successful using nodal explants for propagation on Murashige and Skoog (MS) medium supplemented with various concentrations of single cytokinin or in various combination with auxins. We obtained the maximum percentage (97.6 ± 1.45) response with highest number (16.4 ± 0.60) of shoots per explant on MS medium supplemented with 6-benzyladenine (BA) and ??-naphthalene acetic acid (NAA) at concentrations of 5.0 and 0.5 ??M, respectively. Shoot regeneration frequency was optimized by manipulating pH and using various media. MS medium and pH 5.8 was found to be the optimum for maximum regeneration. Nodal explants from in vitro regenerated microshoots too developed shoots, thus making the process recurrent. Shootlets with 4?C5 nodes were utilized for in vitro rooting, and best response was evaluated on MS medium supplemented with 1.0 ??M indole-3-butyric acid (IBA). The well-developed micropropagated plants were acclimatized (97%) successfully within 2 weeks in soilrite and planted ex vitro in normal garden soil, where they grew well without any morphological and genetic variations. The present regeneration process not only favoured the rapid multiplication but also expressed the regeneration capability of micropropagated plants.     

Non-aerated liquid culture promotes shoot organogenesis in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill

Eucalyptus is very recalcitrant to in vitro culture. In this research, an efficient shoot organogenesis system was developed using 60-day-old plants of Eucalyptus globulus grown in vitro and non-aerated liquid medium to improve shoot proliferation. Cultures were initiated with hypocotyls and leaf segments from plantlets cultivated on semisolid ½ MS modified medium supplemented with 4.44 µM 6-Benzyladenine (BA) and 16.1 µM 1-Naphthaleneacetic acid (NAA). Calli were transferred to shoot induction medium, with either 0.5 or 2.7 µM NAA. Shoot multiplication was carried out on 4.44 µM BA + 0.5 µM NAA medium, and semisolid and non-aerated liquid systems were compared for improving shoot proliferation. Rooting of adventitious shoots was evaluated on medium containing NAA or Indole-3-butyric acid -IBA (5 and 16 µM). Callogenesis was obtained from both types of explants, although shoot formation was only obtained from leaf-derived calli. Shoot proliferation on 4.44 µM BA + 0.5 µM NAA resulted in the most shoots/callus. Non-aerated liquid medium was more efficient in promoting shoot multiplication (53.5 shoots/callus) than was semisolid medium (28.5 shoots/callus). Levels of phenolic compounds were significantly reduced in the shoots cultivated in liquid medium. Efficient rooting (76%) was obtained using 16 µM IBA.     

An improvised in vitro vegetative propagation technique for <Emphasis Type="Italic">Bambusa tulda</Emphasis>: influence of season,sterilization and hormones

Season and concentration of sterilizing agents play a significant role for establishment of aseptic in vitro shoot cultures and sprouting of nodal explants from field growing culms of bamboo species. In the present investigation the nodal segment explants of Bambusa tulda Roxb collected in different seasons and treated with various concentrations of HgCl₂ showed significant variation in aseptic culture establishment and bud break. The rainy season (July–August) recorded with highest of 78% aseptic culture establishment whereas autumn recorded with lowest 46%. Summer and winter seasons emerged to be the best period, registering > 60% in vitro bud break. On the other hand, the autumn season had the lowest value for bud break, i.e. 42%. Among different doses of sterilizing agent tried, HgCl₂ 0.1% found to be suitable for maximum aseptic culture establishment (66%) as well as bud break (59%). However, among the interactions, summer season and the dose of 0.1% HgCl₂ exhibited maximum of 73% response for both aseptic culture establishment and bud break. MS medium (liquid) enriched with 5.0 µM BA + 5.0 µM Kn [Kinetin (N6-Furfuryladenine)] with additional supplementation of 100 µM glutamine + 0.1 µM IAA supported a maximum in vitro shoot multiplication of 4.75 fold. The proliferated shoots were successfully rooted on MS medium (liquid) supplemented 40 µM coumarin. The plantlets transferred to the polythene bags showed 98% survival.     

Direct shoot bud regeneration from shoot tip explants of <Emphasis Type="Italic">Enicostema axillare</Emphasis>: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.     

Plant regeneration by somatic embryogenesis from mature seeds of <Emphasis Type="Italic">Magnolia obovata</Emphasis>

We attempted to develop a method for the regeneration of plantlets from mature seeds of medically important Magnolia obovata via the induction of somatic embryogenesis in vitro. We initially cultured halves of mature seeds on either Murashige and Skoog (MS) medium or B₅ medium that contained 0, 1, 5 or 10 μM gibberellic acid (GA₃) for 1 month and then transferred the half-seeds to half-strength MS basal medium or B₅ basal medium for further culture in the absence of GA₃. Proembryogenic masses (PEMs) were observed 1 month after the transfer of the halved mature seeds to the medium without GA₃. The frequency of formation of PEMs was higher (28%) after initial culture in MS basal medium plus 1 μM GA₃ than in other tested media (0 or 4%). Somatic embryos that had been developed from PEMs were cultured on half-strength MS basal medium or B₅ basal medium for completion of maturation and then transferred to fresh aliquots of the same medium for initiation of germination. The frequency of germination, with the formation of normal primary leaves and roots, was above 80%. We transferred the somatic embryo-derived plantlets to soil for acclimatization and the plantlets continued to thrive.     

In vitro clonal propagation of Gardenia latifolia Ait.: a toy making woody tree

An efficient, in vitro clonal propagation protocol has been established for Gardenia latifolia Ait. using mature nodal explants on Murashige and Skoog (MS) medium fortified with cytokinins (BA/Kn/2-iP) (1.0–5.0 mg l^?1) in combination with auxin IAA (0.5 mg l^?1). Maximum bud break (87 %) with shoot number (7.2 ± 0.26) observed on MS medium supplemented with BA (4.0 mg l^?1) and IAA (0.5 mg l^?1). Maximum number of shoots (30 ± 0.46) with shoot length of (0.9 ± 0.03 cm) observed on MS medium supplemented with BA (2.0 mg l^?1), Kn (2.0 mg l^?1) and IAA (0.5 mg l^?1). Further elongation of shoots (3.5 ± 0.06 cm) was achieved on MS medium supplemented with BA (1.0 mg l^?1) and IAA (0.1 mg l^?1). About 70 % of root induction occurred in half-strength MS medium supplemented with IBA (4.0 mg l^?1) in 4–6 weeks. Further elongation of roots with average length (9.0 cm) was achieved in culture bottles containing vermiculite and ¼ strength MS salts. After their partial hardening in these bottles for 30 days they were transferred to pots containing a mixture of soil and vermicompost (1:1) for acclimatization. The acclimatized plantlets were established in the field successfully with 85 % survival rate.     

In vitro regeneration of <Emphasis Type="Italic">Populus tomentosa</Emphasis> from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ‘fasta-3’. A high frequency of callus induction (>97 %) was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.     

Plantlet regeneration of adult <Emphasis Type="Italic">Pinus massoniana</Emphasis> Lamb. trees using explants collected in March and thidiazuron in culture medium

A protocol for micropropagation using nodal explants from mature Pinus massoniana trees has been developed. Time of explant collection is crucial for the initial success of aseptic culture. Explants collected in early March gave the highest percentage of explant survival (64.5%) and shoot-forming percentage (52.3%). Thidiazuron (TDZ) concentration significantly influenced shoot formation; 4 μM TDZ was optimum, with 4.8 shoots produced per explant with a mean length of 7.1 cm after 120 days of culture. Regenerated shoots rooted for 60 days in basic medium with 1 μM NAA were ready for growth in pots. This is the first report on plantlet regeneration in vitro from mature trees of P. massoniana that provides a reliable method for propagating selected elites.     

In vitro flowering of green and albino Dendrocalamus latiflorus

To propagate Dendrocalamus latiflorus, we used in vivo inflorescences to produce calli on Murashige and Skoog basal (MS) medium supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg/l kinetin, 250 mg/l polyvinyl pyrrolidone (PVP), and 1% coconut milk. Multiple shoots were generated on MS medium supplemented with 0.1 mg/l thidiazuron (TDZ). The green plantlets were successfully transferred to soil. Multiple albino shoots also regenerated and were able to proliferate on medium containing cytokinins, especially TDZ. Albino multiple shoots rooted in medium containing α-naphthaleneacetic acid (NAA), and callus formation was observed in the presence of 2,4-D and picloram. Green and albino regenerates flowered after 8 months of subculture. The flowering ratio increased to 44% after three treatments in medium containing 1 mg/l TDZ. Morphological observations revealed that the in vitro green and albino flower organs were normal. However, pollen derived from the in vitro flowers of both the green and albino plants were sterile.     

Shoot multiplication of <Emphasis Type="Italic">Syringa reticulata</Emphasis> var. <Emphasis Type="Italic">mandshurica</Emphasis> from in vitro cultured seedlings

We developed a shoot multiplication protocol for Syringa reticulata Blume var. mandshurica Hara from in vitro cultured seedlings that derived from in vitro germinated seeds. The shoots could be induced on Murashige and Skoog (MS) medium with proper plant growth regulator combinations of 6-benzylaminopurine (BA) and indole-3-butyric acid (IBA). The better medium for shoot multiplication and growth was MS + 5 mg L^?1 BA + 0.5 mg L^?1 IBA + 20 g L^?1 sucrose + 7 g L^?1 agar, and the corresponding shoot induction rate was 75 %. The plantlets grew well after rooting on 1/2MS medium (macro-elements of MS medium are at half-strength) supplemented with 1 mg L^?1 IBA, and the survival percentage was >80 % at 16 weeks after transplanting.     

苹果叶片再生体系建立研究

以乔纳金苹果试管苗叶片为外植体诱导不定芽再生，在培养基中添加不同浓度TDZ与NAA或IAA配合，使用琼脂或Polygel作为固化剂。结果表明，较适宜的叶片再生不定芽的培养基为TDZ 2．0mg／L和NAA 1．0mg／L，或TDZ 2．0mg／L与IAA 4．0mg／L。较适宜的组培固化剂为5．0g／L的Polygel。在不同的组培固化剂中，卡那霉素均能抑制不定芽的发生数量，但琼脂和Polygel效果不同。     

Shoot proliferation and callus regeneration from nodular buds of Drepanostachyum luodianense

This research reports on an efficient shoot proliferation and callus regeneration system for bamboo.Young, semi-lignified branches with one lateral bud from Drepanostachyum luodianense(Yi et R. S. Wang) Keng f.were used as explants. Disinfection with 0.1% HgCl_2 for 8 min was the optimum treatment and the best medium for bud initiation was Murashige and Skoog(MS) medium containing 3.0 mg L~(-1)6-benzyladenine(BA). Multiple shoots were induced from nodal shoot segments on MS medium containing 5.0 mg L~(-1) BA, 0.5 mg L~(-1) kinetin(Kin), and 1.0 mg L~(-1) naphthaleneacetic acid(NAA). The highest frequency of callus formation(65.6%) was on MS medium containing 4.0 mg L~(-1)2,4-dichlorophenoxyacetic acid(2, 4-D), 0.5 mg L~(-1) NAA, and 0.1 mg L~(-1) thidiazuron(TDZ). The optimum medium for callus proliferation was MS medium with 4 mg L~(-1)2,4-D, 0.5 mg L~(-1) TDZ and 0.5 mg L~(-1) NAA, and the optimum hormone combination was 4 mg L~(-1) BA ? 0.5 mg L~(-1) NAA for callus redifferentiation(up to 85.6%). A 100% rooting was achieved on MS medium supplemented with 2.0 mg L~(-1) NAA and 0.5 mg L~(-1)3-indole butyric acid(IBA). Rooted plantlets were acclimatized in a greenhouse in humus soil ? perlite(1:1) substrate. These micropropagated callus induction and regeneration systems for bamboo will be useful for genetic engineering and multiplication.     

华富苹果离体叶片再生的主要影响因素研究

以花药培养选育的富士品种"华富"组培苗的叶片为试材,研究了离体叶片诱导再生不定芽过程中不同激素种类与组合、暗处理时间等因素对不定芽再生率的影响。结果表明:试管苗继代培养3～4代,取顶部嫩叶3～4片,剪除叶片边缘,叶片以近轴面接种到最佳诱导再生培养基MS+TDZ 2.0mg/L+IAA 0.75mg/L+蔗糖30g/L,pH值5.8,黑暗培养10～15d后,转入光照培养条件下,再生率达83.3%～86.7%。再生不定芽分化15～20d后转接到增殖培养基MS+BA 0.5mg/L+IBA 0.1mg/L上,当不定芽长度约2cm时再转接到生根培养基1/2MS+IAA 1.0mg/L+蔗糖2%,15d后,生根率可达95.0%以上。     

Modulation of in vitro morphogenesis in nodal segments of Salix tetrasperma Roxb. through the use of TDZ, different media types and culture regimes

A comparative performance of two different media formulations (woody plant medium (WPM) and Murashige and Skoog??s (MS) medium) for their ability to inflict in vitro shoot development in nodal segments of Salix tetrasperma Roxb. has been carried out. Thidiazuron (TDZ) in various concentrations was used as a supplement to the basal media. Media types, TDZ concentrations, exposure duration and culture regimes played an important role in affecting multiple shoot production. WPM supplemented with 2.5???M TDZ for 4?weeks exposure was found to be the best for maximum (4.53?±?0.27) shoots production in vitro. Transfer to a secondary medium consisting of 6-benzyladenine (1.0???M) and ??-naphthalene acetic acid (0.5???M) enhanced the multiplication rate and by the end of 12?weeks, 20.33?±?0.33 shoots with shoot length, 4.70?±?0.26?cm were produced on WPM. Rooting of the regenerated shoots was achieved on half strength basal media (either WPM or MS) containing 0.5???M indole-3-butyric acid. In all the experiments, different growth parameters were scored and WPM was found to be superior to MS medium. The regenerated plantlets were successfully acclimatized in the field with about 81?% survival.     

孔雀草杂交F1代的植株再生

以孔雀草（Tagetes patula）子叶、下胚轴和叶片为外植体,通过器官直接发生途径诱导形成不定芽,探讨植物生长调节剂组合、AgNO3浓度、蔗糖浓度和外植体类型等因素对植株再生的影响,建立了再生体系。结果表明：MS＋6-BA 1.0 · L^- 1＋NAA 0.5 · L^- 1＋AgNO31.0 · L^- 1＋蔗糖40 g·L^-1培养基最适合不定芽的分化和增殖,子叶不定芽分化率达90%以上,平均每外植体分化不定芽数达5.3个。不定芽较适生根培养基为1/2MS＋IAA 0.2 · L^- 1＋NAA 0.1 · L^- 1,生根率达到90%。