Somatic embryogenesis of two new <Emphasis Type="Italic">Panax ginseng</Emphasis> cultivars,Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Enhanced somatic single embryo formation by plasmolyzing pretreatment from cultured ginseng cotyledons

Cotyledon explants of Panax ginseng zygotic embryos directly produced somatic embryos on Murashige and Skoog medium without growth regulators. Somatic embryos were formed only near the proximal excised region of cotyledons. Multiple and/or single embryos were formed and the frequency of these formations differed according to the degree of maturity of the zygotic embryos used as the explant source. When cotyledon explants pre-plasmolysed (1.0 M sucrose for 24 h), the frequency of single embryo formation was enhanced regardless of cotyledon maturity. In addition, the distribution pattern of somatic embryos changed markedly because the embryos were formed over the whole surface of the cotyledons. Histological observation revealed that plasmolyzing pretreatment broke the plasmodesmatal connection between cells and when the embryogenic cell divisions commenced, plasmodesmatal strands were hardly observed except for newly formed cell walls. This indicates that the enhanced single embryo formation over the entire surfaces of cotyledon explants might be the result of an interruption of cell–cell interaction by plasmolyzing pretreatment.     

Plant regeneration via adventitious bud formation from cotyledon explants of Panax ginseng C. A. Meyer

Cotyledon explants of ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on medium without growth regulators, with 89% of the explants forming somatic embryos. Cytokinin treatment greatly suppressed somatic embryo formation but stimulated the direct formation of adventitious buds. BAP treatment was more effective than the kinetin treatment for adventitious bud formation. Auxin (0.05 mg/l IBA) in combination with cytokinin enhanced adventitious bud formation, with the highest frequency, 40%, at 0.05 mg/l IBA and 5 mg/l BAP. Adventitious buds were mainly formed near the distal portion of the cotyledons, while somatic embryos were formed near the proximal excised margins. Shoots were developed from adventitious buds after transfer to MS medium with 10 mg/l GA₃. Root formation from the shoots was obtained after the shoots were transferred to half-strength MS medium with auxin (IAA). When the plants derived from adventitious buds were transferred to greenhouse soil, 36% were successfully acclimatized. Received: 7 November 1997 / Revision received: 12 January 1998 / Accepted: 7 February 1998     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Somatic embryogenesis and plantlet regeneration in American ginseng (Panax quinquefolium L.)

Summary Somatic embryogenesis in American ginseng (Panax quinquefolium L.) was investigated from three explant sources (root, leaf and epicotyl) with Murashige and Skoog (MS) medium containing different growth regulators. Mature roots and leaves obtained from 3- to 5-yr-old field-grown plants, and seedling leaves and epicotyls from plantlets grownin vitro, were evaluated. From root and epicotyl explants, callus development was optimal with 3,6-dichloro-o-anisic acid (dicamba) (9.0 μM) and kinetin (KN) (5.0 μM) as the growth regulators. When these calluses were transferred after 3 mo. to dicamba alone (9.0 μM), somatic embryo formation was observed at an average frequency of 15.6% in root explants after an additional 3 mo., and 2% in epicotyl explants after an additional 6 mo. No plantlets were recovered because the embryos germinated to form shoots with no roots. From leaf explants, callus growth was optimal with α-naphthaleneacetic acid (NAA) at 10.0 μM and 2,4-dichlorophenoxyacetic acid (2,4-D) at 9.0 μM. Somatic embryos developed on this medium, with the highest frequency (40%) obtained after 3 mo. from seedling-leaf explants. Calluses on mature leaves formed somatic embryos after 7 mo. with NAA/2,4-D at an average frequency of 30%. Transfer of these somatic embryos to 6-benzyladenine/gibberellic acid (4.4/2.9 μM) promoted shoot development but no roots were observed. Up to 100% of germination was observed within 6 wk on half-strength MS salts containing activated charcoal (1%) and on NAA/2,4-D (5.0/4.5 μM) with charcoal (1%). On the latter medium, somatic embryos enlarged and frequently gave rise to new somatic embryos after a brief callusing phase. The embryos germinated through a two-stage process, involving the elongation of the root followed by the formation of a shoot. The highest recovery of ginseng plantlets from germinated embryos was 61.0%. Following transfer to potting medium and maintenance under conditions of high humidity and low light intensity, the plantlets elongated and developed new leaves. A high percentage (50%) of these plants have been acclimatized to soil.     

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA₃ (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.     

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.     

极东锦鸡儿幼胚子叶的体细胞胚胎发生和植株再生

以极东锦鸡儿未成熟合子胚子叶为外植体进行其体细胞胚胎发生和植株再生研究。在添加不同BA与NAA或2,4-D,外加500mg·L~（-1）水解酪蛋白、30g·L~（-1）蔗糖和8g·L~（-1）琼脂的MS培养基上诱导产生了体细胞胚。在5mg·L~（-1）NAA＋5mg·L~（-1）BA和5mg·L~（-1）2,4-D＋1mg·L~（-1）BA处理中体胚诱导率分别为14%和10%;NAA处理每外植体上诱导出的体胚数量最多为4.3个,而2,4-D为10.5个。体细胞胚经成熟培养后,在添加0.01mg·L~（-1）NAA、20g·L~（-1）蔗糖和6g·L~（-1）琼脂的MS培养基上萌发率达到58.94%。萌发的体胚在MS培养基上长成正常小植株,再生率为87%。经炼苗后的体胚苗移植到草炭土：蛭石：珍珠岩=5：4：1（V/V/V）的栽培基质中,可以正常生长,移栽成活率为40%。     

Developmental and structural aspects of somatic embryos formed on medium containing 2,3,5-triiodobenzoic acid

Cotyledon explants of ginseng (Panax ginseng C.A. Meyer) zygotic embryos produced somatic embryos at a high rate (68%) on medium without any growth regulators. Under this culture condition, apparent polar somatic embryogenesis occurred near the basal-excised portion of the cotyledons. When the cotyledon explants were cultured on medium containing 2,3,5-triiodobenzoic acid (TIBA), an auxin polar-transport inhibitor, the frequency of somatic embryo formation markedly decreased and was completely inhibited on medium containing 20 μM TIBA. On medium containing 5–10 μM, somatic embryos developed sporadically on the surface of the cotyledons and had a normal embryo axis but jar-shaped cotyledons. Embryos with jar-shaped cotyledons were also observed to occur at a high frequency when the early globular embryos formed on hormone-free medium were transferred to medium containing 20 μM TIBA. From these results, it was deduced that endogenous auxin in the cotyledon explants plays an important role in the induction of somatic embryos and that the cotyledon development in somatic embryos is also related to the polar transport of endogenous auxin. Received: 11 October 1996 / Revised version received: 8 January 1997 / Accepted: 26 January 1997     

Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp)

Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 μm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 August 1997     

Shoot Organogenesis from Immature Zygotic Embryo Cultures of Ginkgo biloba

Immature zygotic embryos were cultured on Murashige and Skoog's medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), benzyladenine (BA) and zeatin or with various concentrations of 2,4-D alone. The maximum number (8 per embryo) of adventitious buds formed from cotyledons of heart stage embryos cultured on MS medium with 1 mg dm⁻³ BA and 0.01 mg dm⁻³ NAA. The adventitious buds originated from procambial strands of immature embryo cotyledons and then developed into adventitious bud primordia within 20 d. Adventitious buds transferred to hormone free MS medium grew into shoots, but did not produce plantlets because the shoots failed to root. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plantlet regeneration in Cornus florida

Somatic embryos were initiated from 12 to 15 weeks postanthesis (WPA) zygotic embryos of Cornus florida L. (flowering dogwood) cultured on Murashige-Skoog (MS) or Schenk and Hildebrandt (SH) medium amended with either 3 mg/L 2,4-D or 5 mg/L 2,4-D and 1 mg/L kinetin. White, opaque globular and early cotyledonary stage embryos were formed directly on detached cotyledons from 2 of the 5 trees sampled after 7 weeks of culture. Morphologically mature embryos developed after an additional 4 weeks incubation on medium without growth regulators; however, many of the embryos were fused in pairs along the entire length of the hypocotyl-radicle axis. Indirect embryogenesis was observed from callus cultures initiated from 9 to 15 WPA zygotic embryos. These cultures have continued to produce embryos for 16 months. Many of the embryos formed roots on germination medium, but only 12% formed plantlets and none developed past the first true leaf stage.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - FPA Formalin-propionic acid-ethanol (50%) - WPA weeks post-anthesis     

Somatic embryogenesis,plant regeneration,and the evaluation of camptothecin content in <Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

Summary Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.     

Micropropagation of sweet orange, Citrus sinensis Osbeck. for the development of nucellar seedlings

Protocol for micropropagation of elite plants of sweet orange (Citrus sinensis) through nucellar embryo culture has been standardized. Three to four nucellar embryos and a zygotic embryo could be excised from a single mature seed and successfully generated as healthy plants in basal MS medium. MS medium supplemented with NAA (1 mg/L) or 2, 4.D (1 mg/L) promoted callus development in both nucellar and zygotic embryos. GA3 (1 mg/L) enriched medium induced plantlets initiation but their growth was very poor. No significant differences were observed between initial growth patterns of nucellar and zygotic seedlings developing from the same ovule. Five to six shoots were obtained from collar region of both category of embryos in MS medium supplemented with BAP (1 mg/L) within 60 days of inoculation. The number of plantlets were almost doubled after their transfer in the same medium and culture for another 30 days. Higher doses of BAP resulted in initiation of callus directly from the embryos. The regenerated shoots (2-3 cm) could be rooted in MS medium supplemented with either only NAA (0.75 mg/L) or NAA (0.50 mg/L) and IBA (2.0 mg/L). A number of plantlets could be obtained from a nucellar embryo grown shoot within a limited time period.     

High-frequency regeneration and transformation ofRaphanus savus

We have achieved high-frequency shoot regeneration in radish(Raphanus sativus). Cotyledon explants from four-day-old seedlings were suitable for the effective induction of shoots on Murashige and Skoog’s (MS) medium containing 3.0 mg/L kinetin. We also determined that it was essential to include 1- to 2-ram petiole segments with the cotyledons for efficient induction. When the regenerated shoots were transferred to an MS liquid medium containing 0.1 mg/L NAA, roots formed within four weeks, and normal plant development ensued. We established a transformation protocol using anAgrobacterium binary vector that carries the GUS reporter gene. Preculturing the explants for I d in an MS medium containing 3.0 mg/L kinetin also increased efficiency. Five days of cocultivation proved best for delivering T-DNA into radish. Transformation frequencies of up to 52% were obtained in shoot induction media that contained 3.0 mg/L kinetin.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

Plant regeneration from cotyledonary explants of jute, Corchorus capsularis L.

A liquid culture protocol was developed to regenerate shoots from cotyledons of germinating seeds of jute (Corchorus capsularis L.). Reproducibility of the protocol was tested in three genotypes of jute. Highest bud differentiation rates into normal shoots (via shoot-forming explants) were obtained on modified Murashige and Skoog's liquid medium containing 2.7 μM α-naphthaleneacetic acid and 4.4 μM benzylaminopurine. Regenerated shoots were excised, and the best root formation could be induced in medium with 2.5 μM indole-3-butyric acid and 1.5% sucrose. Bud primordia were formed directly on the cut surface of the cotyledons. Scanning electron micrographs and histological studies confirmed the organogenic nature of the regenerated shoots. The physical condition of the culture medium and the age of the explants played crucial roles in the induction of shoot development using shoots; 2-day-old explants being optimal. Approximately 70% of the shoots were successfully established in soil after hardening. Received: 20 October 1997 / Revision received: 4 October 1998 / Accepted: 27 October 1998