Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination

Commercial deployment of clonal trees via somatic embryogenesis (SE) could increase forest productivity over conventional tree breeding techniques. However, some technical advances need to be made to use SE in clonal forestry with Pinus radiata. For example, the conversion of embryonal mass (EM) into plants is at present a major bottleneck. For this reason, maturation experiments were carried out to determine the effect of the initial amount of EM, activated charcoal (AC) and the best combination of abscisic acid (ABA), sucrose and amino acid concentration in the maturation medium. Germination was evaluated on different media formulations with and without AC. When 100 mg of EM were suspended in liquid medium without AC, cotyledonary somatic embryos were obtained in all the maturation media tested. Maturation medium supplemented with 60 μM ABA, 6% sucrose, and embryo development medium amino acid mixture produced the highest number of cotyledonary somatic embryos, between 10 and 1,550 embryos per gram of EM fresh weight. Approximately half of the tested 25 lines produced more than 600 embryos per gFW. Embryo development was the best when somatic embryos were germinated in half strength modified Quoirin and Lepoivre medium supplemented with 2 g L⁻¹ AC. This protocol simplified and improved SE maturation and germination due to the elimination of subcultures, the large number of somatic embryos obtained from a very low amount of EM, and the elimination of pre-germination treatments, resulting in a significant saving of cost and labor.     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

Factors Affecting Somatic Embryogenesis from Cotyledonary Explants of Safflower

Frequency of safflower (Carthamus tinctorius L.) somatic embryogenesis, number of somatic embryos per responding explant and somatic embryo maturation and germination were affected by genotype, explant age, carbon source, and ethylene. Among 8 cultivars tested, 7 were embryogenic with varying frequencies. The best response was obtained with cv. Girna. Whole cotyledonary explant from 10-d-old plants was best responding compared to 5- or 15-d-old ones. Among different carbon sources, sucrose at 87.6 mM concentration was most suitable for embryo induction, maturation and germination. Of the different ethylene inhibitors, silver nitrate at 50 [micro ]M concentration significantly increased the embryogenic frequency and also the number of embryos per responding explant. Silver nitrate has pronounced effect on embryo maturation but had no effect on germination.     

Callus induction and high-efficiency plant regeneration via somatic embryogenesis in <Emphasis Type="Italic">Papaver nudicaule</Emphasis> L., an ornamental medicinal plant

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 0.1 mg l⁻¹ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg l⁻¹ GA₃ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2–94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

     

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.     

High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Regeneration of plant by somatic embryogenesis in <Emphasis Type="Italic">Pinus rigida</Emphasis>×<Emphasis Type="Italic">P. taeda</Emphasis>

Zygotic embryos at different developmental stages were tested for their potential in the initiation of embryogenic suspensor mass (ESM) lines using immature seeds of Pinus rigida × P. taeda. The highest frequency (1.1%) of ESM was obtained with explants from cones collected on July 1. All excised embryos of the July 1 collection were at the early proembryo stage. Two different culture media were compared. Forty-eight ESM lines were initiated on Pinus taeda basal medium (P6) (0.97%) with 13.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA). However, only four ESM were obtained on a modified Murashige and Skoog medium (MSG; 0.55%). Most of the ESM arose from the seeds that were at the stages ranging from late cleavage polyembryony to the early staged proembryo. Out of 52 lines (0.46%) that were produced from 11,388 explants, only two viable lines (0.018%) (PRT11 and PRT28) survived. As for somatic embryo maturation, the highest number (224/g⁻¹ FW) of matured cotyledonary somatic embryos (line PRT 28) was obtained on a medium containing 100 μM abscisic acid (ABA), 0.2 M maltose, and 1.2% gellan gum. For germination of the somatic embryos, the cotyledonary somatic embryos derived from maturation medium were transferred on half-strength Litvay medium (LM) plus 0.4% gellan gum. The germination rates were high (71.4–96.3%) regardless of the concentrations of either ABA or gellan gum in the maturation medium. Approximately 500 somatic plants were recovered from the germination medium and transferred to the green house; finally most of them were transplanted successfully to the experimental field.     

Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.)

Summary A culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine.     

Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L.

Summary Maturation of somatic embryos of Pinus strobus L. was evaluated on media containing various types (agars and gellan gum), brands and concentrations of gelling agents in the presence of 80 μM ABA and 0.09 M sucrose. The media were characterized with respect to gel strength, water potential and water availability. Embryogenic tissue and somatic embryos cultured on medium with various concentrations of gellan gum were used to determine their water potential (Ψ). Regardless of the type of gelling agent used, gel strength increased with gelling agent concentration and was critical to the maturation response. High gel strength was associated with reduced water availability from the medium to the cultures. The water potential of gelled maturation medium remained constant between 0.4 and 1.0% gellan gum. It is concluded that the embryogenic tissue was exposed to varying amounts of water at the onset of and during the culture period, and that the amount of water in the culture environment in turn influenced the maturation response. Cotyledonary somatic embryos derived from gellan gum medium of high gel strength had a lower Ψ than somatic embryos matured on medium of lower gel strength. Once somatic embryos developed to the cotyledonary stage on the maturation medium, they were transferred to the germination medium. The germination frequency and the number of morphologically normal germinants were higher for somatic embryos matured on medium of high gel strength. Raising the concentration of the gelling agent in the maturation medium may be an alternative to the use of solutes to restrict water available to the embryogenic cultures.     

Somatic embryogenesis in mature zygotic embryos of <Emphasis Type="Italic">Picea likiangensis</Emphasis>

Somatic embryogenesis (SE) was successfully induced from mature zygotic embryos of seven families of Picea likiangensis (Franch.) Pritz after 20 weeks culture on initiation medium. Three basal media (one-half strength LM medium, one-half strength LP medium and improved LP medium) with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (6-BA) were tested but only one-half strength LM medium supplemented with 2,4-D and 6-BA was successful for the embryogenic cultures (EC) initiation. The initiation frequencies of EC varied greatly from different families when culturing on the same initiation medium. The highest frequency (41.3%) was induced from one of the families on one-half strength LM medium supplemented with 3 mg L⁻¹ 2,4-D and 1.5 mg L⁻¹ 6-BA and 16.83% on average for seven families. EC were subcultured and proliferated on the same medium as the initiation one every 10 days. 3 lines of EC induced from the same family were applied in maturation experiment. Cotyledonary somatic embryos were observed after EC were transferred to maturation media of one-half strength LM medium containing 20-80 mg L⁻¹ abscisic acid and 7.5% polyethylene glycol (PEG-4000). However, one-half strength LM medium supplemented with 40 mg L⁻¹ or 60 mg L⁻¹ ABA and 7.5% PEG gave the best maturation and the 3 lines showed different ability in maturation. Over 80% cotyledonary somatic embryos germinated normally on DCR medium containing 0.2% activated carbon. The success on SE induction of the species has provided an effective clonal propagation method for this important tree’s genetic improvement.     

Influence of plant growth regulators,carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock `Colt' (<Emphasis Type="Italic">Prunus avium</Emphasis> × <Emphasis Type="Italic">P. pseudocerasus</Emphasis>)

The frequency of long-term secondary somatic embryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of 4 mg l^–1 of kinetin and 2% of maltose under illumination stimulated shoot development and, subsequently, whole plants have been recovered by applying 1.5 mg l^–1 kinetin to the rooting medium. Although numerous treatments have been tested involving both embryogenic masses and whole embryos, normal embryo germination was observed sporadically. Cold treatment was effective in stimulating secondary somatic embryogenesis with embryo development to the cotyledonary stage, but did not promote their germination. Similarly, a higher concentration (44–55 mg l^–1) of chelated iron than that commonly used in tissue culture media (36.7 mg l^–1) produced, after 3 weeks in culture, almost a 50% increase in the number of embryos at the cotyledonary stage per embryogenic mass. Among the cytokinins tested, 1 mg l^–1 of 6-benzylaminopurine and 0.1 mg l^–1 of thidiazuron were effective in inducing secondary somatic embryogenesis; however, each of them expressed highest efficiency with specific medium and environmental conditions. Furthermore, application of 1 mg l ^–1 thidiazuron reverted morphogenic callus to non-morphogenic callus, particularly in medium containing 2% sucrose. Finally, hormone free medium with 2% maltose enhanced maturation of the emb-ryos to the normal cotyledonary stage. This paper has improved knowledge of embryo culture and plant production in this important genotype, opening the way for genetic improvement by biotechnological techniques, mainly with the aim of modifying the growth pattern of the canopy of sweet cherry grafted on it.     

Optimization of horse chestnut (<Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.) somatic embryo conversion

Factors affecting conversion of horse chestnut (A. hippocastanum L.) somatic embryos into plantlets were evaluated. Anther filament derived embryogenic tissue developed bipolar structures with two cotyledons and a well-developed shoot and root apical meristem upon auxin omittance from the culturing medium. The impact of carbohydrate type (glucose, fructose, sucrose and maltose) and concentration (3 and 6%) on somatic embryo maturation and conversion were evaluated. Although conversion frequencies were high for all treatments, overall quality of regenerated plantlets was poor. Increasing the carbohydrate concentration in the maturation medium did not increase conversion of somatic embryos or quality of regenerated plantlets in terms of shoot height. On the contrary, addition of PEG (polyethylene glycol) in maturation media had a beneficial effect on shoot quality of regenerated plantlets. Sucrose was a superior carbon source when PEG was included in the maturation medium, in terms of conversion rate (65.7%) as well as of shoot quality of plantlets (43.8% of plantlets had shoots >2 cm). Clonal fidelity of the different development stages of somatic embryogenesis and of converted plantlets was assessed by flow cytometry and no major ploidy changes were found.     

Somatic embryogenesis of <Emphasis Type="Italic">Myrciaria aureana</Emphasis> (Brazilian grape tree)

The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation. For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl-l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process.     

Increase in permeability to oxygen and in oxygen uptake of soybean nodules under limiting phosphorus nutrition

In vitro cultures and endogenous abscisic acid (ABA) analyses with gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) were used to study the effects of AgNO₃ and polyethylene glycol (PEG) on white spruce ( Picea glauca ) somatic embryo maturation and endogenous ABA contents, Normally, in the absence of ABA, white spruce somatic embryos cannot mature. However, AgNO₃ and PEG stimulated somatic embryo maturation by increasing the number of cotyledonary embryos. A combination of 100 μ M AgNO₃ and 40 g l⁻¹ PEG was the treatment that was most effective in enhancing cotyledonary embryo formation without exogenous ABA. Either AgNO₃ or PEG was able to increase endogenous ABA levels of the embryogenic culture but a combination of AgNO₃ and PEG was the most effective treatment. Stimulation of cotyledonary embryo formation by AgNO₃ occurred only at low ABA levels, while PEG promoted embryo formation at all exogenous ABA concentrations. Germination tests indicated that AgNO₃ had no negative effects on embryo germination and conversion while the PEG-treated embryos failed to germinate.     

Factors influencing induction and maintenance of <Emphasis Type="Italic">Vitis rotundifolia</Emphasis> Michx. embryogenic cultures

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of ‘Supreme’ and unopened leaves and petioles of ‘Delicious’. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of ‘Alachua’ and ‘Carlos’ cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in ‘Carlos’ (379.3) followed by ‘Alachua’ (350.0) and ‘Delicious’ (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 μM Benzyladenine (BA). Although high embryo germination rates (80–100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6–47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.     

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l⁻¹) and BA (1.5 mg l⁻¹). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources