Culture Of Zygotic Embryos Research Articles

Somatic embryogenesis, micropropagation and plant regeneration of "Early Mature" walnut trees (Juglans regia) that flower in vitro.

Some walnut trees (Juglans regia L.) originating from central Asia display an early flowering phenotype. These "Early Mature" (EM) trees may produce flowers within months of germination. Secondary flowering waves are also observed within a growing season. Inflorescences may carry male, female and hermaphrodite flowers. Progeny obtained from selected EM trees were cultured in vitro to initiate clonal propagation of these genotypes. Embryogenic lines were established through the culture of immature zygotic embryos. Microshoot lines were obtained from germinated somatic or zygotic embryos. Plants showing EM phenotypes were recovered through direct conversion of somatic embryos or adventitious rooting of microcuttings. During the in vitro propagation phase, flower buds were observed on microshoots after three to six subcultures. Histological analysis showed that most of these flowers were hermaphrodite. In vitro apical buds were used to clone the walnut orthologous cDNAs of the AGAMOUS and APETALA 3 MADS-box genes. Northern blots revealed a preferential expression of both of these homeotic genes in flowers. The results highlight the usefulness of EM lines to study the genetic cues controlling flowering and sexual maturity in woody perennials.

In vitro morphogenesis in zygotic embryo cultures of neem (Azadirachta indica A. Juss.).

Immature zygotic embryo cultures of neem yielded highly regenerative cultures, with the response varying with the embryo stage at culture. Early dicotyledonous stage embryos were the most responsive followed by torpedo stage embryos. The embryo cultures differentiated three types of regenerants: somatic embryos (SEs), shoot buds and neomorphs. SEs exhibited morphological abnormalities such as pluricotyledony, fusion of cotyledons and absence of cotyledons. Although these SEs showed secondary embryogenesis, the occurrence of normal dicotyledonous embryos was extremely rare. On MS basal medium 3% of SEs developed a long tap root but a plumular shoot did not appear. However, it was possible to regenerate plantlets from immature zygotic embryo cultures of neem via neomorph formation and adventitious shoot bud formation. The transplantation survival of these plants was more than 80%.

In vitro culture from mature seeds of Passiflora species

The genus Passiflora comprises hundred species, mainly native of the South American tropics and rainforests, which are grouped into 21 subgenera. Some species are widely studied for their economic importance and are chiefly cultivated for production of fruit juice. To obtain a continuous source of material for a screening of secondary metabolites, zygotic embryo culture was attempted for 62 Passiflora species, starting from seeds mainly collected in the wild. Twenty nine of these species produced calli, which had very different growth rates. Plants were successfully regenerated from calli of 13 different species. For 25 of the responsive species this is the first report of in vitro culture.

One hundred years of zygotic embryo culture investigations

Isolation of zygotic embryos from seeds and their culture in a defined medium, initiated by Hanning in 1904, has proved to be a promising method to study the factors that control growth and differentiation of embryos. Using this technique, several investigations have focused on the carbohydrate and nitrogen nutrition during germination of cultured seed embryos and on the effects of plant hormones on their morphogenesis. Culture of immature embryos leads to their germination into weak seeldings, skipping the later stages of embryogenesis, by a process known as precocious germination. Progressively smaller embryos have been cultured by supplementation of the medium with coconut milk or hormonal additives or by osmotic adjustment of the medium by high concentrations of sucrose or mannitol. Although methods have not been developed for large-scale isolation and culture of zygotes, zygotes of maize isolated from embryo sacs and those obtained by in vitro fertilization have been grown in culture into full-term embryos. Embryo culture techniques are widely used to rescue embryos from seed of wide crosses which usually abort and to overcome dormancy of recalcitrant seeds.

DESIGN AND ANALYSIS OF COCONUT EMBRYO CULTURE EXPERIMENTS

The experimental design and data analysis procedures followed for standardizing the CPCRI protocol of coconut zygotic embryo culture are described. The availability of human resources (for initiating the cultures as well as periodic sub-culturing), laboratory space for maintaining culture, infrastructure for preparation of adequate culture media etc, are the important factors to be considered at the planning stage. Since the response of interest per experimental unit is discrete (germinated or not), it is desired to have a “plot size” of 20. Based on the experimental errors obtained for various treatments, attempts were made to obtain the replication size for a specified value of treatment difference to be tested. In most of the experiments, per cent germination was not following the assumption of ANOVA , even after transformation. In such situation, the chi-square test was employed to study the influence of treatments on germination. The coefficient of variation in most of the experiments was below 20 %.

High frequency somatic embryogenesis and plant regeneration in zygotic embryo cultures of Japanese honeysuckle

Japanese honeysuckle plant (Lonicera japonica Thunb.) is rich in iridoid secologanin and is a potentially useful model for the study of secologanin biosynthesis. Culture conditions for high frequency plant regeneration via somatic embryogenesis from zygotic embryo cultures and zygotic embryo-derived embryogenic cell suspension cultures of this species are described. Mature zygotic embryos formed embryogenic calluses at a frequency of 46.7% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichloro-phenoxyacetic acid (2,4-D). Cell suspension cultures were established with embryogenic calluses using liquid MS medium with 4.52 μM 2,4-D. Upon plating onto MS basal medium, embryogenic cell suspension cultures produced numerous somatic embryos, which subsequently developed into plantlets at a frequency of 68%. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 μM 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 μM 2,4-D and 4.44 μM BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.

In Vitro Culture of Zygotic Embryos of Ilex Species

Plants of Ilex argentina L., I. brasiliensis (S.) L., I. brevicuspis R., I. dumosa R., I. integerrima (V.C.) L., I. microdonta R., I. pseudoboxus R., and I. theezans C.M. were obtained by immature embryo culture. Heart-stage zygotic embryos were removed from immature fruits and cultured aseptically on quarter-strength Murashige and Skoog medium with 3% sucrose, 0.65% agar, and 0.1 mg·L-1 zeatin. Cultures were incubated at 27±2°C for 4 weeks, in darkness and subsequently transferred to a culture room with a 14-hour photoperiod (116 μmol·m-2·s-1) for another 4 weeks. Seedlings with two leaves, derived from germinated embryos, were successfully transplanted to pots containing 1 peat: 1 perlite: 1 sand (v/v) and were maintained in greenhouse conditions. From 95% to 100% of transplanted seedlings survived. Chemical name used: 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).

Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana

A simple, rapid and effective system to regenerate Arabidopsis plants via direct somatic embryogenesis has been established. Somatic embryogenesis was induced directly during culture of immature zygotic embryos. The frequency of somatic embryogenesis was strongly influenced by the stage of development of the explants. Explants in different developmental stages were cultured on B5 agar medium containing 5 μM 2,4-dichlorophenoxyacetic acid and the highest frequency (up to 90%) of somatic embryogenesis was observed in zygotic embryos with fully-developed cotyledons. The first somatic embryos developing directly from explant tissue were noticed after 8 days of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all six genotypes tested (Columbia, C-24, RLD, Wassilewskaja, Landsberg erecta and Wilna). Subculture of somatic embryos onto auxin-free medium resulted in their conversion into plants with an average frequency of 79.5%. The regenerates showed normal morphological characteristics and were fertile. All 56 analysed plants displayed a diploid number of chromosomes and two out of 96 (2.1%) tested plants carried a chlorophyll or embryo-lethal mutation.

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or α-naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA3), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.

In vitro zygotic embryo culture of wild Musa acuminata ssp. malaccensis and factors affecting germination and seedling growth

In vitro zygotic embryo culture of wild banana significantly increased the germination compared to greenhouse grown seeds. Embryo orientation and BAP concentration significantly affected germination rate. These factors together with gelling agent, dark and light conditions and coconut water, also showed variable effects on the number of roots per plant, root length, shoot length, number of days to root emergence and number of days to shoot emergence.

692 Plant Embryo Culture Techniques and Applications

Traditional and biotechnological breeding techniques are being united to develop exciting new plants and to improve existing cultivated plants by introducing natural variability from germplasm resources. Intervarietal, interspecific and intergeneric crosses can be accomplished by using plant embryo culture techniques, sometimes also referred to as embryo rescue. Embryo culture involves the isolation and growth of immature or mature zygotic embryos under sterile conditions on an aseptic nutrient medium with the goal of obtaining a viable plant. The technique depends on isolating the embryo without injury, formulating a suitable nutrient medium, and inducing continued embryogenic growth and seedling formation. The culture of immature embryos is used to rescue embryos from hybrid crosses that were once thought to be incompatible because they would normally abort or not undergo the progressive sequence of ontogeny. The culture of mature embryos from ripened seeds is used to eliminate seed germination inhibitors, to overcome dormancy restrictions, or to shorten the breeding cycle. New and exciting cultivars of Alstroemeria, also known as Lily-of-the-Incas, Inca Lily, or Peruvian Lily, have been bred by using zygotic embryo culture; these techniques and applications will be discussed.

Immature embryos culture in Italian red chicory

Chicory (Cichorium intybus L.) embryo maturation in vitro was achieved in B5 medium without growth regulators. Immature embryos were collected at different developmental stages 4–168 h after pollination. Only a few plantlets were obtained from ovules collected shortly after pollination since the embryos at these early stages developed abnormal leaves and roots which regenerated shoots ‘via’ organogenesis. Zygotic embryos collected 24–72 h after pollination needed one month of culture in vitro before acclimation. Embryos collected at the heart or early torpedo stage of development (three days after pollination) produced green plantlets after two weeks of in vitro culture and thus they could be transferred to the greenhouse for acclimation. Culture of zygotic embryos in vitro has shown to be a suitable technique to accelerate the breeding process in biennial cultivated Italian red chicory. In fact it is possible to obtain plantlets ready for performing the selection for the desired agronomic traits one month after pollination. Since flowering occurs after vernalization, the selection can be performed in the correct season before flowering.

Comparative Study Of Anthraquinones From Embryogenic Callus Tissue And Zygotic Embryos Of Frangula Alnus And Rhamnus Catharticus

Organogenesis and somatic embryogenesis were achieved in cotyledon cultures of Frangula alnus Mill. (alder buckthorn) and Rhamnus catharticus L. (common buckthorn), and in zygotic embryo cultures of F. alnus. Histological examination of cotyledonary segment cultures revealed that adventitious buds and somatic embryos arose indirectly through an intervening callus phase. In comparison to zygotic embryos, embriogenic callus of Frangula alnus contained significantly more anthraquinones in the form of free aglycones and different participation of chrysophanol and physcion within synthesized complex of metabolites. Also, in comparison to shoots, root cultures and in particular undifferentiated callus of Frangula alnus, embryogenic callus contained more chysophanol and aloe emodin and less physcion derivatives. Production of physcion derivatives was significantly increased in embryogenic calluses of R. catharticus compared to zygotic embryos. On the other hand, the emodin content was lower than in zygotic embryos. In comparison to shoot, root cultures and in particular undifferentiated callus of common buckthorn, embryogenic callus contained significantly more derivatives of physcion and emodin. The content of chrysophanol and its derivatives was decreased proportionally.

ZYGOTIC EMBRYO CULTURE OF TAXUS CHINESIS VAR. MAIREI AND PLANT REGENERATION THROUGH ORGANOGENESIS

When different media were compared for their effect on zygotic embryo culture, DCR was found more effective than MS medium. From these embryos, adventitious bud induction was achieved by optimizing formular conditions as described in this paper. Adventitious buds were formed from calli derived from hypocotyls of germinated embryos. Finally, adventitious roots were induced and complete plantlets were obtained.

Organogenesis from megagametophyte and zygotic embryo explants of the gymnosperm Dioon edule Lindley (Zamiaceae, Cycadales)

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l−1 glutamine, 100 mg l−1 arginine, 100 mg l−1 asparagine, 60 g l−1 sucrose, 8 g l−1 Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated.

Somatic embryogenesis and plant regeneration in zygotic embryo cultures of balloon flower

Mature zygotic embryos of balloon flower (Platycodon grandiflorum) formed embryogenic calluses at a frequency of 43% when cultured on Murashige and Skoog medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Cell suspension cultures were established from embryogenic calluses using MS liquid medium with 4.52 μM 2,4-D. Following transfer to solid MS basal medium, cell suspension cultures gave rise to somatic embryos, which then developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity.

CHANGES IN PHOTOSYNTHETIC PARAMETERS DURING IN VITRO GROWTH AND SUBSEQUENT ACCLIMATIZATION OF COCONUT (COCOS NUCIFERA L.) ZYGOTIC EMBRYOS

In coconut, #in vitro# development and acclimatization of plantlets still constitute the major bottlenecks for the implementation of tissue culture protocols. In the present paper, the photosynthetic status of #in vitro# grown plantlets resulting from zygotic embryo culture is investigated, combining various complementary approaches, both #in vitro# and #in planta#. Patterns of chlorophyll fluorescence were similar in #in vitro# grown coconut plantlets (aspire: phi MAX= 0.72 and aspire: phi = 0.45) and in autotrophic adult palms (aspire: phi MAX= 0.76 and aspire: phi = 0.50), whereas the chlorophyll content was lower in #in vitro#-cultured plantlets(0.92 mg.g-1 FW) than in autotrophic plants (2.43 mg.g-1 FW). The photosynthetic rate (1.14 micro-mol CO2 m-2s-1) was two times lower in #in vitro# grown plantlets, while transpiration rates were similar in bath #in vitro#-cultured plantlets and in autotrophic palms. Changes in the PEPC: Rubisco carboxylase ratio during the development of #in vitro# grown zygotic embryos (from 89.17 to 0.04 micro-mol CO2 h1mg1rsp) reflected an early transition towards a RubisCO-mediated mode of CO2 fixation. The RubisCO content (172.8 mg.g-1) measured in #in vitro#-cultured plantlets was lower than in autotrophic palms (217.6 mg.g-1). During the acclimatisation phase, the #in vitro# grown coconut plantlets showed a faster decrease in their PEPC: RubisCO ratio than the seedlings, suggesting that an earlier transition from a heterotrophic to an autotrophic mode of carbon fixation takes place in the #in vitro#-derived material. Just before acclimatization, the RubisCO activity in #in vitro# derived plantlets was lower than that in seedlings of the same age. After acclimatisation, RubisCO activities were comparable in both #in vitro# and# in planta# germinated material. #In vitro# grown plantlets displayed several photosynthetic characteristics (aspire: phiMAX, aspire: phi, PEPC: RubisCO ratio and transpiration rates) similar to those of acclimatized plants. These results suggest an early establishment of a photosynthetic metabolism during the #in vitro# development of coconutplantlets. (Resume d'auteur)

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L−1). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L−1) and NAA (0.1 mg L−1). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L−1). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.