A comprehensive review of in planta stable transformation strategies.

Plant transformation remains a major bottleneck to the improvement of plant science, both on fundamental and practical levels. The recalcitrant nature of most commercial and minor crops to genetic transformation slows scientific progress for a large range of crops that are essential for food security on a global scale. Over the years, novel stable transformation strategies loosely grouped under the term "in planta" have been proposed and validated in a large number of model (e.g. Arabidopsis and rice), major (e.g. wheat and soybean) and minor (e.g. chickpea and lablab bean) species. The in planta approach is revolutionary as it is considered genotype-independent, technically simple (i.e. devoid of or with minimal tissue culture steps), affordable, and easy to implement in a broad range of experimental settings. In this article, we reviewed and categorized over 300 research articles, patents, theses, and videos demonstrating the applicability of different in planta transformation strategies in 105 different genera across 139 plant species. To support this review process, we propose a classification system for the in planta techniques based on five categories and a new nomenclature for more than 30 different in planta techniques. In complement to this, we clarified some grey areas regarding the in planta conceptual framework and provided insights regarding the past, current, and future scientific impacts of these techniques. To support the diffusion of this concept across the community, this review article will serve as an introductory point for an online compendium about in planta transformation strategies that will be available to all scientists. By expanding our knowledge about in planta transformation, we can find innovative approaches to unlock the full potential of plants, support the growth of scientific knowledge, and stimulate an equitable development of plant research in all countries and institutions.

In vitro propagation for conservation and genetic fidelity of the near threatened Dimocarpus longan plant.

BACKGROUND: Dimocarpus longan is a tropical tree that produces edible fruit. It is a neglected plant species that is listed as near threatened. In spite of its economic value, the propagation of longan cultivar using conventional methods is extremely difficult. The goal of this research is to produce and conserve this plant through in vitro propagation. RESULTS: In order to form new shoots, sterilized shoot tip explants were cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) or 2-isobentenyl-adenine (2ip). For direct organogenesis, young leaves of new shoots were cultured on MS medium fortified with various concentrations of Thidiazuron (TDZ) or 6-(4-Hydroxy-3-methylbut-2-enylamino purine) (Zeatin). Gibbrellic acid (GA3) at different levels alone or in combination was used for shoot elongation. Also, indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA) were used for root formation. MS medium supplemented with 1.00 mg/l 2ip was suitable for inducing axillary shoots from shoot tips (4.0 axillary shoots/explant). The highest significant 76% and numbers of adventitious buds from leaf base were achieved on MS medium containing 1.0 mg/l TDZ. These buds developed into the longest plantlets on GA3 at 3.0 mg/l and rooted well in ½MS containing 1.50 mg/l IBA plus 0.50 mg/l (NAA). About 70% in vitro plants were successfully acclimatized. The AFLP profile illustrated the genetic stability of gene expression action. The amplified fragment length polymorphisms (AFLPs) profile illustrated the progenies were extremely similar to the mother plants. According to our findings, MS medium containing 25 ppm salicylic acid (SA) and 5 ppm methyl jasmonate (MeJA) produced the highest percentage of apigenin in longan calli (77.09 and 2.637%, w/w). CONCLUSION: A successful and efficient micropropagation protocol has been developed and described here for the first time, and it will be very useful for the clonal propagation and conservation of the near-threatened Dimocarpus longan plant. Micropropagated plants are genetically identical to the donor plant using the AFLP technique. The usefulness of salicylic acid and methyl jasmonate as elicitors for increasing in vitro production of secondary metabolites in plants is demonstrated in this work.

High-frequency adventitious shoot organogenesis from in vitro stem explants of Scutellaria araxensis Grossh.

The present study introduced an in vitro shoot organogenesis protocol for the medicinal plant Scutellaria araxensis (Lamiaceae). Stem, leaf, and petiole explants were cultured in half-strength Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP) alone or in combination with thidiazuron (TDZ), indole-3-butyric acid (IBA), or α-naphthalene acetic acid. Callus formation occurred from stem and petiole explants in most cultures; however, in leaf explants, it was observed only in cultures containing 0.5 mg/l BAP supplemented with TDZ at all concentrations. The highest frequency of indirect shoot induction (100 and 90%) with an average of 20.33 and 12 shoots per explant was observed in stem-derived calli cultured on half-strength MS medium containing 2.0 mg/l BAP plus 0.5 and 1.5 mg/l TDZ, respectively. The best direct shoot organogenesis (40%) was observed in stem explants cultured on half-strength MS medium containing 0.5 mg/l BAP and 0.5 mg/l IBA with a mean of 18 shoots per stem explant. The regenerated micro-shoots were elongated on a medium fortified with 0.5 mg/l gibberellic acid and then successfully rooted in half-strength MS medium supplemented with 0.5 mg/l IBA. The obtained plantlets were acclimatized in a growth chamber with a survival rate of 100%. This study is the first report of a simple and efficient in vitro shoot organogenesis and regeneration protocol for S. araxensis by using stem explants, which could be useful for the conservation, genetic manipulation, and exploitation of biological molecules of this valuable genetic source.

Micropropagación de Calibanus hookeri (lem.) trel (1911). Una especie amenazada / Micropropagation of Calibanus hookeri (lem.) trel (1911). A threatened species

RESUMEN La especie Calibanus hookerii perteneciente a la familia Asparagaceae, está registrada en la NOM-059-SEMARNAT-2010 catalogada como planta amenazada. Sus poblaciones naturales se han visto reducidas de manera importante debido a una explotación excesiva y destrucción de su hábitat, por lo que se requiere de métodos de propagación eficaz que aseguren su conservación. La propagación in vitro es una alternativa viable para especies vegetales amenazadas. En la presente investigación se reporta el protocolo para la micropropagación de Calibanus hookerii mediante la germinación de semilla sin testa inoculada en medio MS complementado con 2.5, 5.0 y 7.0 mg L-1 de 6 benciladenina (BA), cinetina (K), 2-isopentil-adenina (2iP) y tidiazuron (TDZ). Las variables a medir fueron porcentaje de germinación, número y longitud de brotes producidos por semilla. El tratamiento más eficiente fue de 5.0 mg L-1 de BA produciéndose un promedio de 26 brotes por semilla; el tratamiento menos eficaz fue con 2.5 mg L-1 K en el cual solamente se obtuvieron dos brotes por semilla. De las tres concentraciones de 2iP solamente en la concentración de 7 mg. L-1 mostró resultados produciendo 6 brotes por semilla. En lo que respecta a la longitud del brote ningún tratamiento superó al testigo (8.07cm). La eficiencia la germinación in vitro fue de 56-97%.

ABSTRACT The species Calibanus hookerii belonging to the family Aspagaceae is registered in the NOM-059-SEMARNAT-2010 cataloged in danger of extinction and therefore is necessary of propagation methods that assure its conservation and its propagation. In vitro propagation is a viable alternative for endangered plant species. The present investigation reports the protocol for the micropropagation of Calibanus hookerii. This was achieved by seed germination without test in MS medium supplemented with 2.5, 5.0 and 7.0 mg L-1 of 6-benzyladenine (BA), kinetin (K), 2-isopentyl-adenine (2iP) and tidiazuron (TDZ). The variables to be measured were percentage of germination, number and length of shoots produced by seed. The most efficient treatment was 5 mg L-1 of BA producing an average of 26 shoots per seed, the worst treatment was with 2.5 K only produced 2 shoots per seed, of the four cytokinins used 2ip treatment in only one study of the Performed showed results (7 mg L-1) producing 6 shoots per seed. Regarding the length of the shoot, no treatment exceeded the control (8.07cm). Finally, in vitro germination was high (56-97%) in all treatments.

In vitro propagation from axillary buds of the endangered tree Dalbergia congestiflora Pittier (Fabaceae).

Dalbergia congestiflora Pittier is a woody plant species grown in Mexico and Central America and widely used as timber wood and medicinal material. Since D. congestiflora is an endangered species, an in-vitro micropropagation technique is needed for mass propagation of D. congestiflora plantlets. Nodal segments of D. congestiflora stem cuttings grown in greenhouse conditions were disinfected with an appropriate protocol and in vitro established on Murashige and Skoog medium (MS) supplemented with 0.05 mg l-1 benzylaminopurine (BA). The explants showed 10% contamination with 90% survival, and the initial shoot was regenerated in 90% of them. Axillary buds of 45-day-old initial shoots were cultured on MS containing BA (0, 0.05, 0.1, 0.5, 1, 1.5 and 2 mg l-1) singly or in combination with α-naphthaleneacetic acid (NAA) (0, 0.1, 0.5 and 1 mg l-1). A higher shoot number (9.6 shoots/explant) was obtained on MS with 1 mg l-1 BA and 0.1 mg l-1 NAA. Rooting was investigated using half-strength MS, 2% sucrose and different concentrations of indole butyric acid (IBA) (0, 0.1, 0.5 and 1 mg l-1). After 30 days of culture, developing shoots were elongated and rooted in culture medium without IBA, with production of 3.2 roots/shoot. Micropropagated plantlets of D. congestiflora were successfully transplanted and acclimatized to a mixture of peat moss and perlite (2 : 1) with 100% relative humidity in greenhouse conditions with 80% survival at 30 days of culture. This micropropagation protocol will contribute to the conservation of D. congestiflora, and assure the mass propagation for sustainable usage of this species.

An efficient protocol for in vitro direct shoot organogenesis of Sesamum indicum L. using cotyledon as explant.

Establishment of a suitable regeneration protocol is a pre-requisite to carry out transformation study in Sesamum indicum L. (sesame). In this paper, different parameters of regeneration were standardised to develop an efficient protocol for in vitro plant regeneration via direct adventitious shoot organogenesis using de-embryonated cotyledons of sesame as explants. Among the various treatments of MS medium supplemented with 6-benzylaminopurine, thidiazuron and indole-3-acetic acid, maximum regeneration frequency (25.93  ±  2.21%) was obtained in BTI 4 medium (MS supplemented with 33.33 µM BAP with 2.85 µM IAA) within 6 weeks of culture. Regeneration frequency increased further (50.37 ± 2.49%) by fortifying BTI 4 with 29.43 µM silver nitrate (AG 3 medium). Pre-culture of cotyledon explants in AB 3 medium (AG 3 supplemented with 3.78 µM abscisic acid) for 14 days followed by sub-culture in AG 3 medium further improved the regeneration frequency (68.15 ± 2.68%). The highest rate of shoot regeneration (94.82 ± 1.34%) was obtained by pre-culturing 4-day-old cotyledon in a vertical position in AB 3 medium for 14 days and sub-culturing in AG 3 medium for 4 weeks. Regenerated shoots proliferated in MS medium supplemented with 4.44 µM BAP and 1.44 µM gibberelic acid (GA3). The highest frequency (65.33 ± 3.78%) of root induction was achieved by culturing the elongated shoots in MS medium supplemented with 2.69 µM α-naphthalene acetic acid (NAA) for 6 weeks. Rooted plants were acclimatised in soilrite and transferred to soil after 6-8 weeks. The rate of acclimatisation of plants was 76%.

High frequency direct shoot organogenesis of leaf explants and a comparative evaluation of phytochemicals, antioxidant potential of wild vs. in vitro plant extracts of Lysimachia laxa.

The present studies were attempted to develop direct shoot organogenesis from in vitro grown leaf explants of Lysimachia laxa and comparative evaluation of phytochemical and antioxidant potential of in vitro raised and wild plants extracts. The fresh leaves of this species are used for deworming gastrointestinal worm infection in traditional medicine. Overexploitation of this species and poor regeneration has led to rapid decline in wild population, therefore, present investigation was attempted to develop an efficient rapid mass propagation protocol for this species. Our result showed significantly (P < 0.05) high adventitious shoot proliferation of 17.21 ± 0.24 number per leaf explants cultured in Murashige and Skoog medium fortified with 1.25 mg L-1 thiadizuron and 1.0 mg L-1 α-naphthalene acetic acid. Further enhancement was achieved through elongation medium fortified with 1.0 mg L-1 6-benzylaminopurine by average shoot number of 31.1 ± 0.80 and length of 5.96 ± 0.13 cm. Murashige and Skoog medium fortified with 0.50 mg L-1 Indole-3-acetic acid showed high rooting induction (100%) with average root number of 11.70 and length 7.35 cm. All rooted plants were successfully acclimatized in greenhouse and transferred to field condition with a survival rate of 97%. The contents of phenolic and flavonoid were higher in in vitro raised plant in compared to wild plant extracts. Antioxidants assay showed high radical scavenging activity of IC50 1.61 ± 0.07 mg dry material and reducing power of 49.79 ± 0.11 mg/g ascorbic acid equivalent by aqueous methanol extracts of in vitro raised 3-months-old plants in compare to the wild plants. The present protocol is a viable option for pharmaceutical or nutraceutical industries for sustainable utilization of L. laxa with enhanced of phytochemical and antioxidant potency which is not reported elsewhere.

Direct Organogenesis from Immature Female Inflorescence of Date Palm by Gradual Reduction of 2,4-D Concentration.

Inflorescences represent an alternative explant source for superior date palm trees, especially those that do not produce offshoots. They provide large numbers of explants free of fungal and bacterial contamination for successful tissue culture initiation. Furthermore, they are characterized by the capacity of plant regeneration within a short time as compared to other explant types. This chapter focuses on the procedures employed for plant regeneration by direct organogenesis using immature female inflorescence explants, including initiation of adventitious buds, differentiation, multiplication, shoot elongation, rooting, and acclimatization. Adding 5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) into the initiation medium and gradually reducing it to 1 and then to 0.5 mg/L in the subsequent 2 subcultures, respectively, are determining factors in direct adventitious bud formation from the inflorescence. Bud differentiation is obtained on MS medium containing 0.25 mg/L kinetin (Kin), 0.25 mg/L benzyladenine (BA), 0.25 mg/L abscisic acid (ABA), 0.1 mg/L naphthaleneacetic acid (NAA), and 0.2 g/L activated charcoal (AC). Regenerated shoots exhibit sufficient root formation on MS medium supplemented with 2 mg/L indole butyric acid (IBA) and 1 mg/L NAA and subsequent survival in the greenhouse.

Optimized Direct Organogenesis from Shoot Tip Explants of Date Palm.

In vitro propagation is an available alternative to produce uniform and good-quality planting material to establish large-scale date palm cultivation in a short time. This study was carried out to achieve organogenesis and multiplication directly from shoot tips without callus formation, thus avoiding any possibility of undesirable genetic variability among the regenerated plants. The shoot tips explants are cultured on Murashige and Skoog (MS) medium supplemented with 1 mg/L naphthaleneacetic acid (NAA), 1 mg/L naphthoxyacetic acid (NOA), 2.5 mg/L benzyladenine (BA), and 2.5 mg/L isopentenyladenine (2iP). Numerous adventitious buds appeared from the shoot tip explants in darkness after six subcultures at 4-week intervals. Vegetative buds pass through three stages: initiation bud formation, vegetative bud differentiation, and shoot bud proliferation. Shoots are transferred onto medium containing low concentrations of growth regulators for shoot multiplication. The organogenesis protocol described herein consists of six steps: initiation of meristematic buds, multiplication, elongation, rooting, pre-acclimatization, and finally plant acclimatization.

Direct Organogenesis and Indirect Somatic Embryogenesis by In Vitro Reversion of Mature Female Floral Buds to a Vegetative State.

This protocol describes in vitro plant regeneration from mature female inflorescence explants of date palm (Phoenix dactylifera L.) by reversion of floral state (reproductive phase) to the vegetative state. The mature female inflorescence (fully developed) is cultured on MS induction medium containing 10 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/L 2-isopentenyladenine (2iP), and 2 mg/L paclobutrazol (PBZ) or 2 mg/L abscisic acid (ABA). The basal part of the petals has meristematic cells, which can be induced to initiate callus or direct shoot formation depending on the plant growth regulator amendments. Callus forms on the induction medium supplemented with PBZ after 12 weeks, whereas it differentiates into somatic embryos on a medium containing 0.1 mg/L naphthaleneacetic acid (NAA). Direct shoots are regenerated on the induction medium amended with ABA after 24 weeks. Procedures for plant regeneration from mature female inflorescence explants are described, and histological changes which occur during the reversion process are presented.

Efficient regeneration and genetic transformation platform applicable to five Musa varieties

Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

Somaclones em batata identificados pelos descritores mínimos de broto / Potato somaclones identified by minimum descriptors sprout

Este estudo objetivou verificar a estabilidade fenotípica das cultivares de batata 'Asterix' e 'Macaca', avaliar o efeito do tipo de explante (organogênese direta e indireta) e do tempo de subcultivo (12 e 70 meses) em meio nutritivo MS sobre a ocorrência de somaclones nas duas cultivares na produção de batata semente, mediante o emprego de sete descritores mínimos de broto. Em 'Asterix' e 'Macaca' ocorreram somaclones em quatro dos sete descritores, contudo, apenas no formato e pubescência da base do broto houve variação, simultaneamente, em ambas. Os dois genótipos são suscetíveis à ocorrência de variação somaclonal. Registrou-se somaclonesnos dois tempos de subcultivo nas duas cultivares. Diferente do amplamente registrado, identificaram-se somaclones em segmentos apicais caulinares e nodais originados de organogênese direta em 'Asterix' e 'Macaca'.

It was examined the phenotypic stability of potato cultivars 'Asterix' and 'Macaca', evaluated the effect of explant type (direct and indirect organogenesis) and subculture time (12 and 70 months) in MS nutrient medium on the occurrence of somaclonal variation in both cultivars in seed potato production through the use of seven minimum descriptors sprout. In 'Asterix' and 'Macaca' somaclones have occurred in four of the seven descriptors, however, only in the shape and in the base of the bud pubescence that somaclonal variation occurred simultaneously in both cultivars. Both genotypes are susceptible to the occurrence of somaclonal variation. It was identify the occurrence of somaclones both at 12 months and 70 months of subculture in both genotypes. Unlike the widely recorded, somaclones were identified in shoot apical segments and nodal segments derived from direct organogenesis in 'Asterix' and 'Macaca'.

Improved regeneration and transformation protocols for three strawberry cultivars.

Strawberry (Fragaria × ananassa) is an economically important soft fruit crop with polyploid genome which makes the breeding of new cultivars difficult. Simple and efficient method for transformation and regeneration is required for cultivars improvement in strawberry. In the present study, adventitious shoot regeneration has been investigated in three cultivated strawberry plants, i.e., Festival, Sweet Charly and Florida via direct organogenesis using the in vitro juvenile leaves as explants. Explants were collected after sub-culturing on a propagation medium composed of MS supplemented with 0.5 mg/l BA; 0.1 mg/l GA3 and 0.1 mg/l IBA. To select the suitable organogenesis, the explants of the three cultivars were cultured on MS medium supplemented with different concentrations of TDZ (1, 2, 3, and 4 mg/l), then incubated at a temperature of 22 °C ± 2. Medium containing 2 mg/l TDZ revealed the best regeneration efficiency with the three cultivars (72% for Festival, and 73% for Sweet Charly and Florida). After 4 weeks, the produced shoots were cultured on MS medium with different concentrations of BA and Kin to enhance shoot elongation. Results showed that the medium containing 1.5 mg/l BA and 0.5 mg/l Kin revealed highest elongation efficiency (88% and 94%) for Festival and Sweet Charly, respectively. On the other hand, medium containing 1.5 mg/l BA and 0.1 mg/l Kin showed highest elongation efficiency (90%) in Florida. Elongated shoots were successfully rooted on MS medium containing 1.5 mg/l NAA. Furthermore, transformation of the two cultivars, Festival and Sweet Charly, has been established via Agrobacterium strain LBA44404 containing the plasmid pISV2678 with gus-intron and bar genes. Three days post co-cultivation, GUS activity was screening using the histochemical assay. The results showed 16% and 18% of the tested plant materials has changed into blue color for Festival and Sweet Charly, respectively. Out of 120 explants only 13 shoots were developed on bialaphos medium for each cultivar, representing 10.8% bialaphos resistant strawberry shoot. The presence of the both genes bar and uid A was detected by PCR and Northern giving a transformation efficiency of 5%.

Optimization of multiple shoot induction and plant regeneration in Indian barley (Hordeum vulgare) cultivars using mature embryos.

Barley is the fourth most important crop in the world. Development of a regeneration system using immature embryos is both time consuming and laborious. The present study was initiated with a view to develop a regeneration system in six genotypes of Indian barley (Hordeum vulgare) cultivars as a prerequisite to transformation. The mature embryos were excised from seeds and cultured on MS medium supplemented with high and low concentrations of cytokinins and auxins respectively. The MS medium containing 3 mg/L N(6)-benzylaminopurine (BA) and 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) was found to be the most effective for multiple shoot formation in HOR7231 cultivar that could produce 12 shoots per explant. The other cultivars HOR4409 and HOR3844 produced a minimum number of adventitious shoots (1.33 and 1.67 respectively) on MS medium supplemented with 1 mg/L BA and 0.3 mg/L 2,4-D. The elongated shoots were separated and successfully rooted on MS medium containing 1 mg/L indole-3-acetic acid (IAA). The response of different barley cultivars was found to be varying with respect to multiple shoot production. This is the first report of multiple shoot induction and plantlet regeneration in Indian cultivar of barley which would be useful for genetic transformation.

In vitro regeneration of Eucalyptus camaldulensis.

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l(-1) benzyl aminopurine (BAP) and 0.1 mg l(-1) naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l(-1) BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l(-1) Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60-148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.

An efficient in vitro plant regeneration of Dipteracanthus prostratus (Poir.) Nees.- a medicinal herb

Objective: This is the first attempt for an efficient plant regeneration protocol through in vitro direct organogenesis for a valuable medicinal plant, Dipteracanthus prostratus using nodal segment. Methods: Multiple shoots were induced from nodal explants cultured on Murashige and Skoog medium supplemented with kinetin (KIN), 6-benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA). Results: Maximum shoot responses (80%) were obtained with kinetin at 1.0 mg-1. The rate of shoot multiplication was maintained in subsequent subculture on similar fresh culture medium. The highest shoot length (3.96cm) was obtained with seventy three percentages of shoots at 0.2 mg-1 NAA along with 1.0 mg-1 kinetin. Maximum length of root (3.63cm) was formed at 0.5 mg-1 IBA with significant responses (80%). Rooted plantlets were then transferred to perforated plastic cups and grown in the green house at 80% survival rate. Conclusions: The highest survival rate was noticed and this plant developmental protocol could be used for large- scale regeneration of D. prostratus.

Meios de cultura no desenvolvimento de ápices caulinares de mamoneira (Ricinus communis L.) in vitro / Culture media on in vitro development of castor bean (Ricinus communis l.) stem tips

A pesquisa da composição do meio de cultura mais adequado à espécie vegetal e ao tipo de explante empregado é o fator de maior relevância da cultura de tecidos. O cultivo de ápice caulinar com recuperação da planta matriz é uma técnica de grande impacto para a propagação de plantas in vitro, regeneração de plantas livres de vírus, conservação de germoplasma e modificação genética. Objetivou-se, neste trabalho, avaliar composições do meio de cultivo para organogênese direta in vitro a partir de ápices caulinares pertencentes à população FCA-UNESP-PB de mamoneira (Ricinus communis L.), com vistas à propagação clonal de genótipos elite. Foram testadas quatro formulações: MS básico (T1), MS modificado 1 (T2), MS modificado 2 (T3) e WPM (T4), em delineamento experimental inteiramente casualizado, com 20 repetições em cada tratamento, sendo a repetição 1 ápice caulinar/frasco. O T3 apresentou-se superior e diferiu significativamente dos outros tratamentos apresentando 35 por cento dos ápices caulinares diferenciados e desenvolvidos; seguiu-se o T2 com 10 por cento e os tratamentos T1 e T4 não apresentaram diferenciação de tecidos. Os resultados permitiram concluir que os balanceamentos de sais minerais nos meios de cultura avaliados, especialmente a relação NO3 / NH4 e ausência de FeSO4.7H2O, indicaram grande influência no desenvolvimento de ápices caulinares de mamoneira.

The research of the culture medium composition suitable for plant species and the type of explant to be used is the most significant factor in plant tissue culture. The culture of stem tips with plant donor recovery is a technique of great impact for the in vitro plant production, regeneration of virus-free plants, germoplasm conservation and genetic modification. The objective of this study was to evaluate culture media composition in vitro on direct organogenesis of the FCA-UNESP-PB population of castor bean (Ricinus communis L.), from stem tips for clonal propagation of elite genotypes. Four formulations were tested: basic MS (T1), modified MS 1 (T2), modified MS 2 (T3) and WPM (T4) in a completely randomized experimental design with 20 replicates pertreatment with 1stem tip/flask. The treatment T3was superior, and differed significantly from the other treatments, with 35 percent of stem tips developed, followed by 10 percent in T2. T1 and T4 showed no plant tissue differentiation. The results allowed to conclude that the balance of mineral salts and culture media evaluated especially the rate NO3 / NH4 and the absence of FeSO4.7H2O showed nigh influence on the development of stem tips of castor bean.