Different Types Of Explants Research Articles

<i>In Vitro</i> Propagation and Cytological Analysis of <i>Boerhaavia diffusa </i>L—An Important Medicinal Plant Species of Bangladesh

An effective and consistent in vitro propagation technique for Boerhaavia diffusa L. was developed using different types of explants and media compositions. Shoot apex and nodal explants of field grown plants were aseptically cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP and Kn). The maximum number of multiple shoot buds (6.65 ± 0.09) obtained from nodal explants on MS medium containing 2.0 mg/l BAP + 0.5 mg/l IAA. Multiple shoot buds underwent rapid elongation (4.24 ± 0.06 cm) on MS media fortified with 3.0 mg/l BAP + 0.5 mg/l NAA. Half strength MS media supplemented with 1.0 mg/l IBA + 0.5 mg/l IAA was better for induction and proliferation (6.42 ± 0.07) of roots and 75% of plantlets were successfully acclimatized to ex vitro condition, exhibiting a normal development. Somatic chromosome number of in vivo and in vitro grown plants were confirmed to be 2n = 26. Chromosome length ranged from 1.40 to 2.43 μm in the mother plants and 1.34 to 2.31 μm for in vitro grown plants. The total form percent (TF%) of mother and in vitro grown plants was 42.91% and 41.16%, respectively. The karyotype formula of in vivo grown plants was 2n = 26 = 4Lsm + 6Msm + 2Mm + 14Sm, whereas that of the in vitro grown plants was 2n = 26 = 8Lsm + 4Msm + 2Mm + 12Sm. The frequency of the chromosome having arm more than 2:1 was 0.08 for in vivo grown plants and 0.15 for in vitro raised plants. Thus, according to Stebbins classification (1971) the karyotype of both plants falls into 2A symmetrical type.

An improved protocol for efficient transformation and regeneration of Setaria italica.

An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5-9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species.

In vitro callus induction and development of Vernonia condensata Baker with embryogenic potential

ABSTRACT Vernonia condensata Baker has been traditionally used in folk medicine for the treatment of several inflammatory and infectious processes. Overexploitation of this plant species has drastically reduced its population in its natural habitat (Cerrado). Therefore, tissue culture tools, such as somatic embryogenesis, can be used as an alternative method for rapid and large-scale plant regeneration. The objectives of this study were to induce callogenesis in Vernonia condensata from different types of explants and to evaluate the structural aspects of the development of pro-embryogenic masses of this species by means of histological analyses. The formation of calli was induced from leaf explants and internodal segments, which were inoculated in EME medium supplemented with 50 g L-1 sucrose, 0.5 g L-1 malt extract and 2.68 μM NAA, plus varying concentrations of BAP (0.00, 2.22, 4.44 or 8.88 μM). After 40 days, the following morphogenetic traits were evaluated: intensity of callus formation, intensity of oxidation, callus texture, and morphogenesis. The calli with embryogenic masses were analyzed by light and scanning electron microscopy. Both types of explants were responsive regarding callogenesis, with the BAP concentration of 4.44 μM promoting the formation of friable calli associated with a larger percentage of calli with embryogenic masses. Cells from leaf explants and internodal segments were able to dedifferentiate and change into embryonic structures.

Responses of Different Explants of Sweet Potato on Modified MS and LS Based Nutrient Media in vitro

A good number of reports on sweet potato (Ipomoea batatas L.) tissue culture using different tissues of various cultivars with varying level of efficiency and reproducibility are available but callus induction and plantlet regeneration are recalcitrant and limited to a few genotypes in response to different treatments in vitro. Reported in this paper is a procedure in which several explants of three high yielding and drought tolerant sweet potato cultivars (SK010, WHCH005 and PRAP496), mostly grown and cultivated in the highlands of Papua New Guinea (PNG) were used to induce embryogenic callus and regenerate plantlets. To achieve a reliable and an efficient system for inducing embryogenic callus on stem, petiole and leaf disc explants, a modified form of Murashige and Skoog (MS) and Linsmaier and Skoog (LS) media, supplemented with 1 gL-1 picloram and 8 gL-1 agar was used. This procedure resulted in large amount of embryogenic calli that were potentially capable of regenerating whole plantlets on all the different types of explants tested. Further attempts made in plantlet regeneration failed, however ways in which improvement of the tested regeneration media can be made for plant regeneration in similar studies were discussed.

The influence of explants type and orientation on growth and development of Mandevilla sanderi (Hemsl.) Woodson in vitro

Mandevilla sanderi is an important commercial ornamental pot plant. Traditional vegetative propagation is limited due to the low rate, therefore there is a need to develop an alternative, more efficient method. There is an interest in development of micropropagation technology for the species, as it allows to obtain a lot of offsprings in a relatively short time. The aim of the present work was to estimate an influence of explants type and position on regeneration of Mandevilla sanderi in tissue culture. Four different types of explants (leafy shoot tips, decapitated leafy shoot tips, defoliated shoot tips, decapitated and defoliated shoot tips) were used in the experiment, which were placed on the media vertically, while defoliated shoot tips were placed horizontally or vertically upside down. The explants were cultivated on a Murashige and Skoog (MS) medium supplemented with 1 mg·dm–3 benzyladenine (BA) and 0.5 mg·dm–3 indole-3-butyric acid (IBA). It was noted that both explants orientation and positioning, influenced the multiplication rate. Defoliated shoot tips placed horizontally were characterized by higher multiplication rate (6.8) in comparison to upside down vertical positioning (3.2). It was also observed that removal of shoot apex improved axillary branching, while defoliation of shoots placed in a normal position reduced multiplication rate.

Callus formation and organogenesis in tissue culture Deschampsia antarctica E. Desv.

Aim. The aim of the work was to determine the optimal conditions for induction and proliferation of tissue culture obtained from D. antarctica plants from various localities of the Maritime Antarctica. Methods. Tissue and organ culture techniques. Results. The media В5 supplemented with 2 mg/l 2,4-D + 0,1 mg/l BAP, В5 supplemented with 10 mg/l 2,4-D + 0,2 mg/l BAP and МС, supplemented with 5 mg/l 2,4-D + 0,1 mg/l Kin were optimal for callus induction from different types of explants. The media with a reduced concentrations of auxins and cytokinins were the most effective for maintenance of continuous tissue culture compared to the media for callus induction: B5 + 2 mg/l 2,4-D mg/l + 0,1 mg/l BAP and MC + 1 mg/l 2,4-D + 0.1 mg/l Kin. Tissues from shoot growth point and leaf explants of genotypes DAR12a and G/D12-2a on medium B5 with 2 mg/l 2,4-D + 0.1 mg/l BAP and B5 with 10 mg/l 2,4-D + 0,2 mg/l BAP demonstrated the ability to spontaneous organogenesis and formed separate shoots. Conclusions. Conditions have been determined for the induction and proliferation of tissue culture from leaf, root, and shoot growth point explants of D. antarctica. The frequency of callus formation depended on the mineral composition of medium, ratios and concentrations of growth regulators, type of explant, and genotype of a donor-plant. As a result of spontaneous organogenesis, regenerated plants were obtained, conditions for their rooting in vitro were elaborated. The proposed methods for induction and proliferation tissue culture of D. antarctica in vitro can be used to produce the plant material useful for a various investigations. Keywords: Deschampsia antarctica E. Desv., tissue culture, organogenesis in vitro, frequency of callogenesis.

MORFOGENESIS ANGGREK (Anoectochilus formosanus) SECARA IN VITRO

ABSTRACT Anoectochilus formosanus is one species of Orchids, known as a “Jewel Orchids” and have been used as a folk medicine in China and Taiwan.The aim of this study was to examine the response of using different types of explants and combination of growth regulators TDZ and BAP to morphogenesis of Anoectochilus formosanus Orchids by in vitro. The results showed that using different types of explants had very significant effect to the percentage of callus and shoot morphogenesis on 12 weeks after planting, the number of adventitious buds, adventitious shoot length, and callus diameter on three weeks until 12 weeks after planting. The best explant to callus morphogenesis was showing on shoot tip explant (50%) and to shoot morphogenesis was showing on auxillary buds and internode explant (100%). The best number of adventitious buds was observed in second internode explant with average number of shoots are 7.62 shoots. The best adventititous length was observed in axillary buds explant with average number of shoot length in 1.16 cm, and the best callus diameter was observed in shoot tip explant with average of diameter in 2.2 mm. The combination of plant growth regulator TDZ and BAP had a very significant effect to adventitious length on 10 weeks after planting, and a significant effect to adventitious length on 11 and 12 weeks after planting. The best adventitious length was observed on 0.25 mg L -1 TDZ + 0.75 mg L -1 BAP in 1.13 cm per explant on 12 weeks after planting. The best combination to callus and shoot morphogenesis was observed on 0.75 mg L -1 TDZ + 0.25 mg L -1 BAP. There was no interaction between used a different types of explant and combination plant growth regulator to morphogenesis Anoectochilus formosanus orchids by in vitro.

In vitro shoot regeneration of mint (Mentha sp. L.) Using different types of explants and levels of benzylaminopurine

An experiment was conducted in the Tissue Culture Laboratory of the Department of Horticulture, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Salna, Gazipur-1706 from March 2013 to February 2014 for in vitro shoot regeneration of mint using different explants and levels of benzylaminopurine (BAP) in full strength MS media. Three different types of explants viz. nodal segment, shoot tip and leaf were evaluated using three levels of BAP (1.0, 2.0 and 3.0 mg/l) along with control for shoot regeneration. Results revealed that shoot tip and nodal segments performed better than leaf as explants in almost all the characters studied. Shoot tip and nodal segments initiated shoot within the shortest time of 9.6 and 10.6 days, respectively with 1.0 mg/l of BAP. Regarding number of shoot per explant and number of node/shoot, shoot tip and nodal segments along with 1.0 mg/l of BAP performed superior at almost all days after inoculation. In case of interaction of explants and BAP, better performance was recorded in most of the studied parameters from shoot tip and nodal segments alongwith 1.0 mg/l BAP. Therefore, for in vitro shoot regeneration of mint, shoot tip or nodal segment may be used with 1.0 mg/l of BAP.Bangladesh J. Agril. Res. 43(4): 703-716, December 2018

TRANSFER OF GLUCANASE GENE TO RESIST LATE BLIGHT DISEASE IN POTATO (Solanum tuberosum L.)

Potato late blight is a pandemic disease caused by the highly virulent (Phytophthora infestans) fungus. The regeneration capacity was done among three tested potato cultivars (Spunta, Diamont and Desiree). Two different types of explants (inter-node and leaf) were cultured for calli induction and plant regeneration .The highest value of leaf explants was for Desiree cultivar (80%), which induced from developed callus in the 2,4-D medium. The best value of maximum shoot regeneration was also for Desiree cultivar in the BA, IAA and GA3 media from leaf which proved to be more effective. Then cloning of glucanase gene in pRI plasmid which carrying kanamycin resistance (nptII) gene was perfomed and followed by transformation in Agrobacterium tumefaciens LBA4404 strain which used for plant transfection. Nucleotide and amino acid sequences of transformed Agrobacterium were analyzed. The putative transgenic plantlets genomes and cDNA of the isolated RNA for glucanase gene.

Micropropagation’s Complete Protocol of Red Araçá (Psidium cattleianum, Myrtaceae) from Germinated Seeds in vitro

Red Araçá’s (Psidium cattleianum) micropropagation processes have shown enormous potential both in terms of research and as a sustainable native resource to be used in the areas of food production, ecology, and pharmacology. Currently, however, despite that potential, research efforts involving this myrtaceae, native to the Brazilian Atlantic Forest, have been scarce. With that in mind, this study set out to establish micropropagation techniques that would allow the development of a feasible protocol to be used with Red Araçá, achieving its mircropropagation from in vitro germinated seeds. Different types of explants were tested for in vitro establishment. For the multiplication of nodal segments, different concentrations of BAP and IAA combinations were tested in an MS medium. Using the same medium, different concentrations of ampicillin were applied in order to determine its influence on the decontamination of the apical segments. The BAP and IAA combinations were also used to test their effects on the in vitro explants’ development and rooting. During pre-acclimatization, survival of in vitro rooted plants was tested in a nebulizer chamber, using a commercial substrate and that same substrate mixed with washed sand (1:1). In essence, it was indeed possible to develop a complete protocol for the micropropagation of the Red Araçá from seedlings obtained by in vitro germination. The in vitro introduction of the Red Araçá was rather efficient, independently of the type of explants used. As the BAP and IAA concentrations increased, so did the in vitro seedlings’ development (7 leaves explant-1) and rooting (67%). Additionally, the in vitro rooted plants exhibited a high rate of survival (80%) in the pre-acclimatization phase, independently of the substrate used.

Protocol for the Micropropagation of Coconut from Plumule Explants.

Coconut is a crop that is economically important in several countries throughout the world. Unfortunately, production is decreasing because palms are affected by very serious phytoplasma diseases, such as lethal yellowing, and also most of coconuts are already very old. On the other hand, markets for coconut products have been rapidly growing in recent years. Hence, replanting of most cultivation surface worldwide, as well as establishing new surface, is urgently needed. This is an immense task, requiring at least a billion coconut palms that cannot be accomplished by traditional propagation through seed. Therefore the biotechnological alternative of micropropagation by somatic embryogenesis is needed. Research has been carried out on this subject in laboratories in several countries studying different approaches, testing different types of explants. The most responsive tissue has been plumule from zygotic embryos. A protocol for micropropagation of coconut based on plumule explants is described here. It involves the use of different media that are based on Y3 medium complemented with activated charcoal, gelling agent, sucrose, and growth regulators. These media allow the formation of embryogenic callus and somatic embryos, growth of shoots, and development of plantlets.

Direct somatic embryogenesis in Epipactis veratrifolia, a temperate terrestrial orchid

ABSTRACTMost temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA3) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L−1) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA3. FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia.

In vitro adventitious organogenesis and histological characterization from mature nodal explants of Citrus limon

An efficient and robust system of shoot organogenesis for lemon has been developed as an initial step in the transformation and in vitro mutagenesis protocols. In vitro adventitious organogenesis was studied and histologically characterized using mature nodal explants of two important cultivars of Citrus limon, ‘Verna 51’ and ‘Fino 49’. The effects of several plant growth regulator concentrations, incubation conditions, basal medium, explant type and position along the shoot, and ethylene inhibitors were determined. The presence of 1–3 mg L−1 N 6-benzyladenine, in combination with 1 mg L−1 gibberellic acid, was essential for the development of adventitious buds, with the optimum concentration depending on the cultivar, while organogenesis was lowest when N 6-benzyladenine was used in the medium without gibberellic acid. When the effect of different basal media was studied, the best results for percentage of responsive explants were obtained with Murashige and Skoog medium for both cultivars, while the number of buds per regenerating explant was less than 2 in Woody Plant medium. Attempts were made to identify the type of explant that had the highest regeneration ability using mature shoots of C. limon. When different types of explants (nodal segments with the buds completely removed or internodal segments) were compared, the highest percentage of responsive explants was obtained with nodal segments, while with internodal segments, the regeneration percentage was very poor. Histological analysis showed that nodal segments were more reactive than internodal ones and those structural changes occurred sooner in nodal segments. The existence of a morphogenetic gradient along the shoot was also observed and nodal segments from the apical region exhibited the highest regeneration ability. An initial culture period of 2–4 wk in the dark, depending on the cultivar, clearly improved the regeneration percentage and the number of buds for lemon adult explants. Silver thiosulphate increased the regeneration percentage in adult explants of both cultivars, and the best results were obtained with 20 μM silver thiosulphate.

Optimization of Conditions for Genetic Transformation and In Vitro Propagation of Artemisia carvifolia Buch

The excessive attention in the use of plants as medicine is credited to the occurrence of active principles whose pharmacological activities have been investigated. Due to the limited production and very less quantity of these important metabolites present in the plant cells, their genetic engineering and increased in vitro production is the point of focus for many years and can be achieved by in vitro transformation and propagation of desired plant. In the current study, we report transformation protocol for Artemisia carvifolia Buch with Agrobacterium tumefaciens C58C1 harboring β-glucuronidase as reporter and neomycin phosphotransferase as selectable marker gene. We have optimized simple regeneration conditions after transformation involving two different types of explants (leaf and stem) on different media formulations for direct organogenesis and best regeneration response. MS media with 2.5 g/L benzylaminopurine (BAP), 0.25 g/L naphthalene acetic acid (NAA), giving maximum number of shoots was selected. Rooting was obtained on ½ MS medium supplemented with NAA (0.1 mg/L). Transient expression of gus reporter gene was observed in the leaf and stem explants after 2 days of bacterial infection. Artemisia carvifolia Buch can be successfully transformed with Agrobacterium tumefaciens strain C58C1 by using controlled in vitro regeneration conditions. Findings of the current study would be useful for micro propagation and genetic transformation of Artemisia carvifolia Buch in future.

Investigation of callogenesis and indirect regeneration of Freesia × hybrida Bailey ‘Argenta’

Abstract An investigation was conducted to study the effects of explant sources, plant growth regulators, carbohydrates and light conditions on indirect cormlet regeneration and the induction of embryogenic callus of freesia (Freesia × hybrida Bailey ‘Argenta’). Sections of two different types of explants, corms and pupae (cold storage-produced corms), were placed on Murashige and Skoog (MS) media containing different concentrations of plant growth regulators. The results showed that the highest percentage of callus induction (100%), the highest callus growth (15 mm diameter) and the best type of calli were achieved for pupa explants grown on the medium that contained 4 mg L−1 1-naphthaleneacetic acid (NAA) and 2 mg L−1 6-benzylaminopurine (BAP) in the dark. Increasing BAP up to 3 to 4.5 mg L−1 resulted in the maximum number of regenerated cormlets from 1 cm2 calli (2 cormlets) under light conditions. Overall, the best rooting of regenerated cormlets was achieved on MS media supplemented with 1 mg L−1 indole-3-butyric acid (IBA). In the next stage, high quality calli were subcultured on MS media containing sorbitol, sucrose, maltose and mannitol (0, 5, 10 and 15 g L−1). The results indicated that 15 g L−1 maltose was able to induce the highest percentage of embryogenic callus, with an average of 88.9% on media containing 2 mg L−1 BAP and 1 mg L−1 NAA.

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana.

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

In vitro regeneration and cardenolide determination of an endemic foxglove, Digitalis cariensis (Aegean Foxglove)

The genus Digitalis L. is one of the most pharmacologically important plant genera because many Digitalis species produce cardenolides, which are commonly used for cardiac insufficiencies. In this study, different pretreatments were employed to increase germination of Digitalis cariensis seeds, which germinate poorly because of extensive dormancy. Seed germination was improved when seeds were pretreated by scarification followed by soaking in sterile distilled water overnight. In addition, the regeneration capacity of four different types of explants (cotyledonary leaf, root, hypocotyl, or flamingo-bill-type [FBT]) was tested on Linsmaier and Skoog (LS), Murashige and Skoog (MS) and Gamborg’s B5 (B5) medium containing no plant growth regulators or supplemented with 0.5 mg L−1 thidiazuron (TDZ) alone or in combination with 0.25 mg L−1 indole-3-acetic acid (IAA). Among the explants tested, only FBT explants regenerated. The highest mean number of shoots was produced on LS medium supplemented with a combination of 0.5 mg L−1 TDZ and 0.25 mg L−1 IAA, producing 3.9 shoots per FBT explant after 4 wk of incubation. Regenerated shoots were rooted on LS medium containing different concentrations of IAA or indole-3-butyric acid (IBA) (0.1, 0.5, or 1.0 mg L−1). The greatest number of roots developed on medium supplemented with 1.0 mg L−1 IAA, which produced 5.6 roots per shoot after 3 wk of culture. Cardenolides were profiled by HPLC analysis from basal leaves of D. cariensis plants from natural populations and from in vitro-derived plantlets. There was no significant difference in lanatoside contents (A, B, and C) between the two sources. Digoxin and digitoxin were not detected in either source.

High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33%), cotyledon (90.11%), leaf (62.96%) and petiole (91.10%) explants on MS medium supplemented with 3.5mg/l BAP + 0.019mg/l NAA 2.5mg/l BAP + 0.5mg/l NAA, 4.0mg/l BAP + 0.5mg/l NAA and 4.5mg/l BAP + 0.019mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10mg/l NAA was found best for root regeneration (100%) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

不同培养基配方对美花石斛生根培养的影响

目的：为美花石斛种苗的规模化生产提供理论依据。方法：以将继代培养的美花石斛长至0.2~0.6 cm左右的丛生芽分割外植体，分别转入不同的培养基中。研究MS培养基浓度，激素(NAA)浓度对美花石斛组培苗生根的影响。结果：MS培养基的浓度对美花石斛组培苗生根影响较大，组培苗在1/2 MS培养基中有明显的促进作用。在培养基中添加激素(NAA)对美花石斛组培苗生根有很大的促进作用，随着激素(NAA)浓度的增加，组培苗生根率越高，当NAA增加到0.7 mg/L时达到86%，再增高会出现生长缓慢，叶变枯黄。结论：当培养基配方为1/2MS + 0.7 mg/L时，美花石斛组培苗生根及生长效果最佳。 Objective: The effect of different culture medium formulas on Dendrobium loddigesii root culture was studied to provide theoretical basis for the standardization production. Method: We investi-gated the effect of media components on the induction of shoot regeneration for different types of explants to research the effect on taking root for Dendrobium loddigesii. Results: The media com-ponents have a great influence on taking root. And 1/2 MS medium can obviously promote rooting. And with the increasing of NAA, the rooting rate rises. But when the NAA increased to 0.7 mg/L, and the rooting rate reached 86% and then increased again, there will be a slow growth with the leaves turning yellow. Conclusion: The best culture medium formula is 1/2 ms + 0.7 mg/L NAA for Dendrobium loddigesii to take root and grow.

An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66%) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5mg/l BAP and 5mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200mg/l) for selection, standardization of cocultivation time (45min) and acetosyringone concentration (150μM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.