Histological analysis of plant regeneration from plumule explants of Cocos nucifera

Plant regeneration was achieved from plumules excised from mature zygotic embryos of a local coconut cultivar (Sri Lanka Tall). A detailed histological study was undertaken to gain a better understanding of the cellular changes that occur during plant regeneration from plumule tissues. This study led to the identification of the cellular origin, specific cell characterization and development pattern of embryogenic calluses. It also revealed that abscisic acid induces plant regeneration through somatic embryogenesis. The presence of incomplete somatic embryos that lacked shoot poles was also observed.     

Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos

Immature zygotic embryos of ginseng produced somatic embryos on MS medium without growth regulators. However, in the culture of mature zygotic embryos, excision of the embryo was required for somatic embryo induction. Somatic embryos formed only on excised cotyledons without an embryo axis or on excised embryos without the plumule and radicle of the axis. This observation suggests that the axis tip of the embryo might suppress somatic embryo production although the cotyledon tissues have predetermined embryogenic competency. To clarify the role of the embryo axis on somatic embryo formation, excised plumules or radicles were placed in direct contact with the basal cut-ends of cotyledons. The adhesion of plumules or radicles highly suppressed somatic embryo formation from cotyledon explants. When an agar block containing exudate from excised plumules or radicles was placed in contact with the cut end of the cotyledon, a similar inhibition was observed. These results suggest that embryogenic competence is suppressed by endogenous inhibitors present in the axis tip of the zygotic embryo.     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5???M). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15?d followed by cycles of 8?h dark and 16?h light, under white fluorescent lamps with a light intensity of 3,000?lm/m² and at temperature of 28?±?2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9?mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.     

鱼腥草体细胞胚胎发生和植株再生

目的:利用鱼腥草的叶片和叶柄为材料,进行体细胞胚胎诱导及植株再生研究。方法:运用正交设计试验,考察在改良的MS固体培养基上添加不同种类、不同浓度的植物生长物质组合及其配比对鱼腥草愈伤组织诱导、体细胞胚胎发生及植株再生的影响。结果:鱼腥草无菌苗叶片在含有2,4-D 1.0 mg/L 6-BA 0.5 mg/L的改良MS培养基上能诱导出胚性愈伤组织;胚性愈伤组织在含有6-BA 1.0 mg/L的改良的MS培养基上诱导体细胞胚的发生;叶柄在含有6-BA 1.0 mg/L改良MS培养基上直接产生体细胞胚。体细胞胚在改良的MS NAA0.1 mg/L 6-BA 1.0 mg/L的培养基上能够快速繁殖,形成大量不定芽,在不加任何激素的MS培养基上就可以萌发出不定根,发育为成完整植株,在MS IBA 1.0 mg/L的固体培养基上能够形成大量的根。结论:建立了鱼腥草体细胞胚胎发生及植株再生的体系。     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

Comparison of different methods for plant regeneration and transformation of the legume Galega orientalis Lam. (goat's rue)

 For the first time, regeneration and transformation have been achieved from the legume Galega orientalis Lam. (goat's rue). Two different regeneration protocols are described, one based on direct shoot induction from meristems and the other involving callus induction and shoot induction from callus with the plant growth regulator thidiazuron (TDZ). Different media and explants were evaluated. Three different transformation methods were compared: cocultivation with four different Agrobacterium tumefaciens strains, electroporation of embryos and apical meristems and particle bombardment of embryos. TDZ-promoted shoot induction on calli from immature embryos gave the best results. Transformation using this regeneration protocol was most successful with particle bombardment. Stable transformation has yet to be proven. Received: 11August 1997 / Revision received: 6 April 1998 / Accepted: 1 March 1999     

Unfertilized ovary: a novel explant for coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) somatic embryogenesis

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 μM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 μM abscisic acid, followed by plant regeneration medium (with 5 μM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.     

Genotypic effects on somatic embryognesis and plant regeneration from callus cultures of alfalfa

Fifty genotypes of each of three cultivars of alfalfa (Medicago spp.) were tested in three medium protocols for their capacity to produce somatic embryos and plantlets from callus cultures. Highly productive genotypes produced somatic embryos regardless of medium protocol or explant source, while other genotypes produced somatic embryos in a medium-specific or explant-specific fashion. The results showed that embryogenesis in mature leaf-derived calli could be predicted from the frequency of embryo formation in cotyledon-derived calli of the same genotype. The results also indicated that highly productive genotypes can be selected from cultivars with a low frequency of regeneration.     

Indirect somatic embryogenesis and morphohistological analysis in <Emphasis Type="Italic">Capsicum chinense</Emphasis>

Capsicum chinense is recalcitrant in in vitro morphogenesis. No efficient, reproducible somatic embryogenesis regeneration system exists for this species, impeding regeneration from transformed cells. An indirect somatic embryogenesis protocol is developed using mature C. chinense zygotic embryo segments (ZES). The ZES cultured in semi-solid Murashige-Skoog (MS) medium supplemented with 8.9 μM naphthaleneacetic acid, 11.4 μM indoleacetic acid and 8.9 μM 6-benzylaminopurine, developed an embryogenic callus and 8% of the calli developed somatic embryos. Torpedo-stage somatic embryos were detached from the callus and subcultured in semi-solid MS medium without growth regulators, producing a 75% conversion rate to plantlets with well-formed root tissue. Histological analysis showed the developed structures to have no vascular connection with the callus and to be bipolar, confirming that this protocol induced formation of viable somatic embryos from mature C. chinense ZES. All acclimated plantlets survived under greenhouse conditions. This protocol will facilitate regeneration of genetically transformed plants using either biolistics or Agrobacterium tumefaciens approach.     

Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants

Summary Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embrogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenenis callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.     

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.     

Regeneration of cassava plants via shoot organogenesis

A novel regeneration system based on direct shoot organogenesis is described for cassava. Plants could be regenerated at high frequency by inducing shoot primordia on explants derived from cotyledons of cassava somatic embryos. After a passage on elongation medium, the regenerated shoots were easily rooted in hormone-free medium and could be successfully transplanted to soil. Using the shoot-organogenesis-based regeneration method, up to eight transplantable plantlets per explant could be regenerated. The system was optimised first for one cassava cultivar, and then its transferability to three other cultivars was demonstrated. This method widens the scope of in vitro regeneration modes of cassava, and is also compatible with Agrobacterium-mediated transformation. To develop an efficient system for production of somatic embryos for regeneration experiments, conditions for inducing primary and cycling somatic embryos were also studied, and highly efficient plant regeneration via germination of somatic embryos was achieved using maltose instead of sucrose in the culture medium, and combining paclobutrazol with 2,4-dichlorophenoxyacetic acid in the embryo induction medium. Received: 25 January 1997 / Revision received: 10 February 1997 / Accepted: 20 February 1997     

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.     

Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble)

The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum. Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1). Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration. At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively). Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12%. Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009. Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations. Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

Plant regeneration through callus initiation from thin mature embryo fragments of wheat

A wheat regeneration system was developed using mature embryos. Embryos were removed from surface-sterilised mature caryopses (winter wheat Odeon cultivar and spring wheat Minaret cultivar) and ground to pieces through a sterile nylon mesh. The fragments were characterised by means of the image analysis technique. They were 500 M mean diameter and most of them were elongated. They were used as explants to initiate embryogenic calli on solid medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid. The morphogenic pathway of the initiated calli was followed for a 40-day culture period. Active cellular division occurred within 24 hours of cultivation. Several hundred calli were produced from 100 fragmented embryos within 3 days. A 90% callus induction rate was achieved and proembryos appeared by the 8th day of culture. The highest embryogenic calli induction rate of 47% was obtained when 2,4-dichlorophenoxyacetic acid was suppressed after a 3–4 week induction period. Two regeneration methods were finally compared. A total of 513 plantlets were produced. The optimal protocol produced 25–30 plants per 100 embryos. This regeneration method may be suitable for transformation applications.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.