Production Of Somatic Embryos Research Articles

Somatic Embryogenesis in Hedychium bousigonianum

An efficient primary somatic embryo (SE) and secondary somatic embryo (SSE) production system was developed for the ornamental ginger Hedychium bousigonianum Pierre ex Gagnepain. Addition of two ethylene inhibitors, salicylic acid (SA) and silver nitrate (AgNO3), to the culture media improved the system. Callus was initiated and proliferated on a medium containing Murashige and Skoog (MS) basal salts supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid and 4.6 μM kinetin. Friable callus was transferred to a liquid medium containing MS basal salts, B5 vitamins, 0.6 μM thidiazuron, and 8.9 μM 6-benzylaminopurine to induce somatic embryogenesis. The effects of various concentrations of SA (0, 25, 50, 75, 100, 125, 150 μM) and AgNO3 (0, 10, 20, 30, 40, 50, 60 μM) on callus growth, SE, and SSE development was further evaluated. The rate of callus growth decreased as the concentrations of SA or AgNO3 increased. AgNO3 and SA at all concentrations stimulated SE and SSE development better than the control although a decrease in embryo production was observed at higher concentrations of both SA and AgNO3. The best concentrations for SA were 75 and 100 μM, whereas for AgNO3, they were 30 to 50 μM for both SE and SSE production.

The association of homeobox gene expression with stem cell formation and morphogenesis in cultured Medicago truncatula.

Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24–48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are “hijacked” for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-009-0988-1) contains supplementary material, which is available to authorized users.

Do stress-related phytohormones, abscisic acid and jasmonic acid play a role in the regulation of Medicago sativa L. somatic embryogenesis?

This study examined the role of endogenous abscisic acid (ABA) and jasmonic acid (JA) in indirect somatic embryogenesis of Medicago sativa L. A multiplex GC-MS/MS technique allowed quantitative single-run analyses of ABA, JA, 12-oxophytodienoic acid (OPDA) and indole-3-acetic acid (IAA). The preparation of initial explants led to a strong accumulation of ABA, JA and OPDA but not of IAA. Substantially higher levels of ABA, JA and OPDA were detected in developing somatic embryos than in callus or embryogenic suspension. Fluridone (FLD) decreased ABA, JA and OPDA levels. Indoprofen (INP) appeared to be a specific inhibitor of octadecanoid biosynthesis. Somatic embryo production and development were negatively affected by FLD or INP. Only INP (0.5 μM) applied during proliferation phase increased the number of cotyledonary embryos. The results strongly indicate the involvement of ABA and JA in somatic embryogenesis of M. sativa. Surprisingly, low IAA contents in comparison to stress-related compounds (ABA, JA and OPDA) were detected in explants, embryogenic tissues and somatic embryos.

Possibility of Sweet Corn Synthetic Seed Production

Somatic embryogenesis in sweet corn has been reported by a number of workers. However, the knowledge maintaining storage life, vigor and viability of these somatic embryos are limited. A model system of synchronous somatic embryos production combined with encapsulation to synthetic seed was studied in sweet corn (Zea mays var. saccharata). In this study immature zygotic embryo cultured on N6 medium, contained 2, 4-D 2 mg L(-1) and sucrose 60 g L(-1) form the embryogenic callus. Higher 2, 4-D levels did not show increasing in inducing embryogenic callus. If the concentration of 2, 4-D decreased globular-stage, somatic zygote form the roots. Somatic embryo develop without surrounding nutritive tissues and protective seed coat has been devoted to causing somatic embryos to functionally mimic embryo, then was encapsulated by 3% (w/v) sodium alginate with 4-6 mm in diameter. It was found that when synthetic seed were treated with 60 g L(-1) sucrose and stored at 15+/-2 degree Celsius for 2 weeks, the survival rate of synthetic seed were 44%, after 8 days of germination test, it was found that there were 91% of which were normal seedling and 9% were abnormal seedling. This result indicated that there is a possibility in sweet corn synthetic seed production. Anyhow, more research for better technique are further required.

Endogenous ethylene in indirect somatic embryogenesis of Medicago sativa L.

Ethylene biosynthesis during different phases of somatic embryogenesis in Medicago sativa L. cv. Rangelander using two regeneration protocols, RPI and RPII, was studied. The highest ethylene production was detected during callus growth on induction medium in both regeneration protocols. Significantly less ethylene was produced by embryogenic suspension than by callus (RPII). Developing embryos synthesized higher amounts of ethylene than mature embryos. Production of ethylene was strongly limited by the availability of 1-aminocyclopropane-1-carboxylic acid and also by ACC-oxidase activity. However, removal of ethylene from culture vessels’ atmosphere using KMnO4 or HgClO4 had no significant effect on callus growth, somatic embryo induction and development. Reducing of ethylene biosynthesis by aminoethoxyvinylglycine substantially decreased somatic embryo production and adversely affected their development, indicating ethylene requirement during proliferation and differentiation but not induction.

음나무 배발생 캘러스의 증식 및 체세포배 발달을 위한 액체 현탁 배양조건 확립

본 실험은 음나무 배발생 세포의 증식 및 체세포배 발달을 위한 액체 현탁 배양조건의 확립을 위해 수행되었다. 배발생 세포의 생장율은 접종 밀도가 증가할수록 감소하였다. 배발생 세포의 증식에 가장 효과적인 접종 밀도는 0.1 g/100 ml 로서 이 농도에서 세포의 생장율이 가장 높았다. 배양기간에 따른 배발생 세포의 생장 패턴 및 세포 주기 (G1, S, G2/M) 분석 결과, 세포의 생장은 배양 5일 후부터 증가가 시작되어 15일 까지 급격히 생장하였으며 그 이후에는 점차 감소하였다. 배양 기간 별 세포 주기 (cell cycle)의 변화가 명확하게 관찰되어 배양 5일째 5기는 초기의 5.5% 에서 11.7%로 두 배정도 증가하였으며, 배양 15일 이후부터는 다시 초기의 세포 주기로 되돌아가면서 안정화되는 것으로 나타났다. 따라서 음나무 배발생 세포의 현탁배양은 15일의 주기로 배양하는 것이 증식에 가장 효과적인 것으로 생각되었다. 한편 배발생 세포에서 체세포배의 유도는 배양초기의 접종 밀도가 중요한 것으로 나타났다. 0.5 g/L 의 낮은 밀도로 접종 시에는 65% 이상의 어뢰형 배가 유도된 반면 접종 밀도가 높아질수록 어뢰형으로의 배발달은 급격히 감소하였다. 초기의 접종 밀도가 증가할수록 특히 어뢰형 배의 발달은 지연되었으나 구형 및 심장형 배의 유도는 초기 접종밀도에 영향을 받지 않았다. 이상의 실험결과로 음나무 액체 배양 시 초기 접종 밀도를 조절함으로써 배발생 캘러스의 증식 및 체세포배를 효과적으로 유도할 수 있었으며 이는 체세포배 생산을 위한 배양 기간의 단축이 가능함을 보여주는 결과이다. This study was conducted to establish the optimal suspension culture system for both the propagation of embryogenic cells (ECs) and the induction of somatic embryos (SEs) of Kalopanax septemlobus. The proliferation rate of ECs was reduced as the inoculum density was increased; the highest rate was obtained when 0.1 g/100 ml of cells was initially inoculated. According to the analysis of cell growth pattern and cell growth cycle (G1, Sand G2/M), the cell growth started in 5 days culture initiation, grew rapidly until 15 days and then decreased gradually. Distinctive changes of the cell growth cycle by the culture periods was also observed; the growth cycle was doubled from initial 5.6% to 11.7% of S stage in 5 days culture and then reached in stable stages again. Therefore, the results indicated that a 15-day-cycle was the optimal culture period for the propagation of the ECs through the suspension culture. Furthermore, the cell inoculum density was also important for the induction of SE; more than 65% of SEs at the torpedo stage was induced by using the low level of cell inoculum (0.5 g/L), while the higher inoculum densities were rapidly reduced the proportion of SEs at that stage. Although the higher inoculum density delayed the development of SE, it did not affect the proportion of SEs at the globular and heart stage. In conclusion, this study showed that the suspension culture of the Kalopanax septemlobus ECs through the control of inoculum density was an efficient way for both the propagation of ECs and the induction of SEs, suggesting that the development of this system might help to reduce the culture period for the somatic embryo production.

In vitro cultivation of donor quince shoots affects subsequent morphogenesis in leaf explants

The effect of in vitro cultivation of donor shoots on subsequent morphogenesis in leaf explants of quince (Cydonia oblonga Mill.) clone BA29 was investigated. Proliferating donor shoots were cultured in ventilated or closed vessels under different photosynthetic photon flux densities (PPFD; 200 and 100 µmol m-2 s-1) with 0, 15, 30 g dm-3 sucrose. Shoots grown in ventilated vessels, especially with sucrose at 15 or 30 g dm-3, were better developed with fully expanded leaves compared to those in standard closed vessels. Leaves collected from pre-treated donor shoots were used to assess regeneration capacity. Somatic embryo production was highest in leaves harvested from shoots cultured in closed vessels with 30 g dm-3 sucrose and in ventilated vessels with 15 and 30 g dm-3 sucrose and under high PPFD which was, in comparison with the control treatment (closed vessel, 30 g dm-3 sucrose and low PPFD), about 2 to 2.5 times higher. A similar response was observed for root regeneration.

Effects of exogenous polyamines and inhibitors of polyamine biosynthesis on endogenous free polyamine contents and the maturation of white spruce somatic embryos

The maturation of somatic embryos is a pivotal stage of somatic embryogenesis process. Thus, this study is aimed at investigating the role of polyamines on the maturation of somatic embryos of white spruce (Picea glauca). Two inhibitors of the biosynthesis of polyamines methylglyoxal bis-(guanylhydrazone) (MGBG) and dicyclohexylamine (DCHA) or three exogenous polyamines (putrescine, spermidine and spermine) were added into a modified HLM-1 maturation medium inoculated with embryogenic tissues. Medium responses were assessed with respect to the production of mature somatic embryos, the cellular concentration of free polyamines and the concentration of 1- aminocyclopropane-1-carboxylic acid (ACC) and N-malonylaminocyclopropane-1-carboxylic acid (mACC), the precursor of ethylene biosynthesis and its conjugate, respectively. Results show that DCHA brought about a significant decrease in the concentration of free cellular putrescine during the first two weeks of maturation, whereas MGBG induced a significant decrease of both spermidine and spermine. Moreover, both inhibitors (1 mM DCHA or 1 mM MGBG) reduced the total number (63 and 52%, respectively) and the number of normal (62 and 54%, respectively) somatic embryos. Addition of 5 mM spermidine or 1 mM spermine increased the total number (94 and 113%, respectively) and the number of normal (134 and 143%, respectively) somatic embryos. Finally, the intracellular concentrations of ACC and mACC significantly increased in the presence of exogenous spermidine while decreasing in the presence of MGBG, suggesting that spermidine interacts with ethylene metabolism. These results clearly demonstrate that polyamines play an important quantitative and qualitative role during the maturation of white spruce somatic embryos.

In Vitro Regeneration of Four Commercial Cotton Cultivars ( Gossypium hirsutum L.) Grown in Xinjiang, China

Genetic improvement of cotton through biotechnology has been limited by lack of an efficient regeneration system. An efficient somatic embryo production and maturation procedure was thus developed to regenerate plantlets from hypocotyls of cotton cultivars Xinluzhong 20, Xinluzao 24, Xinluzao 33, and 03298 grown in Xinjiang. Calli were effectively produced by 0.01 to 0.10 mg L −1 kinetin (KT) and 0.10 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D), with a better result by 0.02 or 0.10 mg L −1 KT and 0.10 mg L −1 2,4-D. Split hypocotyl segments and double amounts of KNO 3 during induction of calli were beneficial to the emerge of embryogenic calli. Embryogenic calli and globular-stage somatic embryos were effectively initiated with high concentration of KT and low concentration of 2,4-D in ECM media (0.05 or 0.10 mg L −1 KT and 0.01 mg L −1 2,4-D). Embryos were further developed into plantlets in MSBF medium under the conditions of dehydration and ventilation, which were achieved using filter paper on medium and cotton tampon sealing flask. Using this protocol, normal plantlets with strong roots were developed from these cotton cultivars in 6 to 8 months. The successful regeneration protocol established in this study can be used to improve cotton cultivars by genetic engineering.

Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants

Alternative procedures for the production of Narcissus L. somatic embryos were investigated. Somatic embryogenesis was initiated on ovary explants isolated from cv. Carlton bulbs, chilled for 12 weeks at 5°C. The explants were cultured on MS media with 3% sucrose and growth regulators: Picloram or 2,4-D (10 or 25 μM) and BA (1 or 5 μM) for 12 weeks in the culture systems: continuous cultivation on solid media, continuous cultivation in liquid media and sequential cultivation using cycles in liquid and solid media. Two types of somatic embryogenesis, indirect and direct, were observed. The developmental pathway depended on the period of exposure to liquid media. Somatic embryos were formed via embryogenic nodular callus on solid media. 2,4-D and BA stimulated the process. The 4-week and 8-week liquid medium treatments resulted in the development of somatic embryos directly from the ovary explant tissue. The highest number of somatic embryos was noted under the influence of 25 μM 2,4-D and 5 μM BA in explants cultivated for 8 weeks in liquid medium and then, for 4 weeks, on solid medium. The effects of inoculum density on biomass increase and the formation of somatic embryos in cultures obtained on a medium with 25 μM 2,4-D and 5 μM BA were also checked. The highest biomass increase was observed after subculturing in liquid medium containing 0.5 μM NAA and 5 μM BA when the density of inoculum was 0.5 g/25 ml of the medium. The highest number of somatic embryos was noted when the density of inoculum was 1.5 g/25 ml.

Influence of in vitro environment on somatic embryogenesis of Coffea arabica L. cv. Caturra rojo: the effects of carbon dioxide on embryogenic cell suspensions

The influence of environment in the culture vessel is a factor that has very little study in the process of somatic embryogenesis. The present research was carried out with the objective to determine the effects of carbon dioxide on somatic embryogenesis of Coffea arabica cv. Caturra rojo. Embryogenic cell suspensions were cultured under different carbon dioxide concentrations (2.5%, 5.0%, and 10.0%) in the gases mixture and two control treatments, one with passive exchange and the other with forced ventilation. The results demonstrated that there were a larger number of somatic embryos formed with a concentration of 2.5% CO2. The differentiation of these somatic embryos of coffee in embryogenic cell suspensions (130 × 103 SE l−1) was also stimulated. The effects of CO2 on somatic embryogenesis were demonstrated when the control with passive exchange was compared with forced ventilation control, because in the former, where there was an accumulation of CO2, the production of somatic embryos was greater. CO2 could stimulate the formation and differentiation of somatic embryos directly, which led to a modification of the pH patterns of the culture medium or indirectly when producing changes in the pH that favored the somatic embryogenesis process.

Expression of WUSCHEL in Coffea canephora causes ectopic morphogenesis and increases somatic embryogenesis

Cell differentiation depends on the proper and sequential expression of key genes required for morphogenesis. Several aspects of control are required for this which include: chromatin modifications, DNA methylation, correct amount of particular transcription factors, proper nuclear arrangement, etc. During the last few years the homeobox transcription factor WUSCHEL (WUS) has been shown to cause dedifferentiation when expressed on somatic cells followed by a production of new stem cells that can lead to somatic embryogenesis or organogenesis. We found that expression of WUS in coffee plants can induce calli formation as well as a 400% increase somatic embryo production. The results show that transgenic expression of the transcription factor WUS can be useful to increase somatic embryogenesis in heterologous systems. However, a critical developmental stage and additional hormonal requirements are required for the induction of embryogenesis by WUS in Coffea canephora.

Coffee Yield and Mineral Cycle in Intercropping of Coffea canephora and Some Species of Timber Shade Trees

Cocoa (Theobroma cacao L.) like most tropical trees is recalcitrant in tissue culture. Somatic embryogenesis is generally efficient micropropagation technique to multiply elite material. However, Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. One of the factors often considered as a component of in vitro recalsitrance is a high phenolic content and oxidation of these compounds. In cocoa tissue culture accumulate large amounts of poliphenolics compounds which probably impair further development. This study was conducted to investigate the composition of phenolic compounds in cocoa flower and leaves, and their changes troughout the somatic embryogenesis process. Calli were induced in cacao floral and leaves explants on a half-strenght Murashige and Skoog medium containing 30 g/L Glucose and combination of 2,4 dichlorophenoxyacetic acid (2,4 D) with kinetin (kin). Total polyphenol content was observed on Sulawesi 1 cocoa clone. Embryogenic and non-embryogenic callus were also compared. The percentage of callus production from flower tissue is 85%, percentage of embryogenic callus 40 %, although the percentage of somatic embryo production from embryogenic callus callus is 70%. The conservation of callus into somatic embryos followed by decline in phenol content and an increase in peroxidase. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. So that, phenolic compound can influence the production of calli and an absence the phenolic compound can enhance production of somatic embryo.Kata kunci: Theobroma cacao L., polifenol, embrio somatik, kalus, flavonoid, katekin, in vitro recalcitance

First Report of Plant Regeneration via Somatic Embryogenesis from Shoot Apex-Derived Callus of Hedychium muluense R.M. Smith

ABSTRACT The genus Hedychium consists of about 50 species, with increasing popularity as ornamentals and potential as medicinal crop plants, but there are no reports on somatic embryogenic regeneration of any member of this genus. The objective of this investigation was to establish an in vitro regeneration system based on somatic embryogenesis for Hedychium muluense R.M. Smith using shoot apex-derived callus. Callus was induced and proliferated on a modified Murashige and Skoog (MS) medium (CIPM) supplemented with 9.05 μM 2–4, D, and 4.6 μM kinetin. Friable callus developed into somatic embryos upon transfer to liquid medium (MS basal salts and Gamborg's vitamins) that was supplemented with 0.6 μM thidiazuron (TDZ) and 8.9 μM 6-benzylaminopurine (BA) and shaken for four weeks. The cultures were then transferred to three Hedychium embryo-development media (HEDM) of varying strengths: HEDM, 1/2 HEDM, and 1/4 HEDM. All three media contained both 0.6 μM TDZ and 8.9 μM BA. Somatic embryo production was higher in full strength HEDM, which produced an average of 103 somatic embryos/explant, half of which could be converted into shoots within a month. Regenerated shoots were readily rooted on a medium supplemented with 0.6 μM 3-indoleacetic acid (IAA) and acclimatized before transfer to the greenhouse.

Calcium-dependent mechanism of somatic embryogenesis in Panax ginseng cell cultures expressing the rolC oncogene

The Panax ginseng 2c3 embryogenic cell culture was earlier obtained by callus cell transformation with Agrobacterium rhizogenes rolC. Calcium channel blockers (LaCl3, verapamil, and niflumic acid) reduced the production of somatic embryos in the 2c3 culture, implicating the Ca2+ signaling system in plant somatic embryogenesis. The protein kinase inhibitors W7 and H7 also decreased the yield of somatic embryos in the 2c3 culture. The total CDPK expression in the 2c3 culture was 1.2-to 1.5-fold lower than in a control callus culture as a result of a silencing of the genes belonging to the PgCDPK1 (PgCDPK1a and PgCDPK1b) and PgCDPK3 (PgCDPK3a) subfamilies. Expression of the PgCDPK2 subfamily genes (PgCDPK2b and PgCDPK2d) was increased. It was assumed that some genes of the PgCDPK1, PgCDPK2, and PgCDPK3 subfamilies were responsible for generation of embryogenic cells in the 2c3 culture. For the first time, rolC expression and embryogenesis were associated with changes in the expression of certain CDPK genes.

Somatic embryo production from immature seeds of carob (Ceratonia siliqua L.): histological evidence

SummaryImmature seeds isolated from pods of carob (Ceratonia siliqua L.) were used as a source of explants for callogenesis and somatic embryogenesis. Suitable developmental Stages of immature seed explants, and in vitro culture conditions for the production of translucent callus were identified. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the culture medium was found to be necessary for callogenesis. Both the developmental Stage of the explant and the 2,4-D concentration had significant effects on the percentage of callus production. A high proportion (67.0 ± 1.0%) of immature seed at Stage 2 gave rise to callus when cultured on a medium containing 9.0 µM 2,4-D, which was significantly higher than all the other treatments tested. Embryogenic calli consisted of a white-translucent mass proliferating all around the explants and gave rise to somatic embryos. On day-45 of culture, transverse sections of callus showed de-differentiated cells located on the surface, which gave rise to pro-embryos. On day-70, early somatic embryos were observed as globular white structures, and others were at the heart-shaped stage. On day-90, complete somatic embryos were initiated which had attained the cotyledonary stage, characterised by a defined meristematic area and separation of a protoderm and pro-vascular bundles.

GENETICALLY STABLE SOMATIC EMBRYOS OF F1 CUCUMBER (CUCUMIS SATIVUS) HYBRIDS

Explants were taken from hypocotyls, cotyledons and leaves of five cucumber cultivars. The maximum callus weight in the induction medium was 1215.4 mg with the cv. Natica using hypocotyl explants and the minimum was 487.6 mg with cv Mascot using leaf explants. When auxin was available in the induction phase somatic embryos were obtained; the absence of cytokinin caused a reduction in the number o: embryos. The number of somatic embryos increased with sucrose concentration to maximum of 9% sucrose. Three weeks on induction medium was needed for produc tion of somatic embryos; prolonged incubation (6 weeks) gave poor somatic embryo numbers. As auxin concentration decreased in maturation medium somatic embryo yield increased; the highest number was obtained in the absence of auxin. Generally the culture of callus on maturation medium in darkness was superior to 16 h light foi somatic embryogenesis. Leaf explants of the cultivar Profito gave 37.3 embryos pei replicate which was the highest number recorded. In addition, this cultivar yieldec embryos in the shortest time period (6 weeks). Satisfactory somatic embryo numbers were obtained after three weeks on induction medium, plus three weeks on maturation medium, or four weeks on induction medium, plus two weeks on maturation medium, or five weeks on induction medium, plus one week on maturation medium Comparison of characteristics of cucumber plants derived from somatic embryogenesis with those obtained from seed showed no significant difference in phenotype ol plants, or fruits, or genotype (using RAPD DNA tests).

Direct somatic embryogenesis from leaves, cotyledons and hypocotyls of Hippophae rhamnoides

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm−3 kinetin and 0.2, 0.5 mg dm−3 indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.

A short history of plant biotechnology

The foundations of modern plant biotechnology can be traced back to the Cell Theory of Schleiden (Arch Anat Physiol Wiss Med (J Muller) 1838:137–176, 1838) and Schwann (Mikroscopische Untersuchungen uber die Ubereinstimmung in der Struktur und dem Wachstum des Tiere und Pflanzen. W Engelmann: Leipzig No 176, 1839), which recognized the cell as the primary unit of all living organisms. The concept of cellular totipotency, which was inherent in the Cell Theory and forms the basis of plant biotechnology, was further elaborated by Haberlandt (Sitzungsber K Preuss Akad Wiss Wien, Math-Naturwiss 111:69–92, 1902), who predicted the production of somatic embryos from vegetative cells. This brief historical account traces the development of technologies for the culture, regeneration and transformation of plants that led to the production of transgenic crops which have become central to the many applications of plant biotechnology, and celebrates the pioneering men and women whose trend-setting contributions made it all possible.

Clonal plant production from self- and cross-pollinated seed families of Pinus sylvestris (L.) through somatic embryogenesis

Several factors affecting somatic embryogenesis (SE) in Pinus sylvestris from self- and cross-pollinated seed families were studied with the aim of producing large quantities of clonal plants. Somatic embryogenesis initiation from zygotic embryos was improved on a medium with lower than standard concentrations of 2,4-dichlorophenoxyacetic acid (2.2 vs. 9.5 μM) and 6-benzyladenine (2.2 vs. 4.5 μM). On this medium, initiation rates of four controlled crosses, including one self-cross, varied from 3% to 25%. Among the maturation factors tested, the concentration of abscisic acid (ABA 80, 120 μM) had no significant effect on the production of mature somatic embryos when the medium contained 0.1 M sucrose. When sucrose concentration was 0.2 M, however, 1.4 times more mature somatic embryos were produced on medium with 80 μM compared with 120 μM ABA. Under our best maturation conditions, mature somatic embryos accumulated amounts of storage proteins that were similar to the amounts in mature zygotic embryos. Activated charcoal exerted a beneficial effect on mature somatic embryo production of 24-week-old cultures; there was no evidence of such an effect in 8-week-old cultures. Thirty-seven embryogenic lines from a self-cross and an out-cross were chosen for clonal plant production. Highly embryogenic lines produced mature somatic embryos that were more likely to convert to plants than those from less embryogenic lines. After 4 months of growth in a shade house, plantlet survival rates exceeded 70% for 31 lines out of 35. This report describes an improved method for accelerated production of large quantities of Scots pine for clonal tests.