Germination Of Somatic Embryos Research Articles

Somatic embryogenesis mediated micropropagation of polyembryonic cultivars of mango Vellaikolumban and Olour using nucellus tissue

Abstract Somatic embryogenesis mediated micropropagation from nucellus tissue was attempted in polyembryonic mango at the ICAR-Indian Institute of Horticultural Research, Bengaluru during 2016–2019. Studies were conducted to optimize the different stages of somatic embryogenesis namely induction, proliferation, conversion, maturation, germination of somatic embryos and ex vitro establishment of plantlets using nucellus tissue. For culture initiation, various developmental stages of fruits ranging from less than 20 days post-pollination to more than 60 days-post pollination of cultivars Vellaikolumban and Olour were utilized. Fruits of 30–40 days post-pollination in Vellaikolumban and 40–50 days post-pollination in Olour gave better percentage callusing with fewer days for callus initiation and callus formation. Embryonic calli developed on induction medium containing Rugini olive (RO) constituents, 6% sucrose, and 5 ppm each of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and Giberellic acid (GA3). Upon shifting to proliferation medium (RO with 6% sucrose, 5 ppm each of 2, 4-D and GA3, 400 mgL−1of glutamine and 20% v/v coconut water) pro-globular and globular embryos were formed within 4–5 weeks. Further development of somatic embryos through early heart, late heart and cotyledonary stages occurred in conversion medium (half strength B5 salts, full RO organics, 5 ppm of Indole-3-Acetic Acid (IAA), 20% coconut water and 200 mgL−1 of casein hydrolysate). Maturation of embryos was obtained by using 0.01 mg L−1of Abscisic acid (ABA) and 100 mg L−1of Polyethylene glycol (PEG). Germination of embryos with shoot and root initiation was observed in the presence of 5 ppm of zeatin. Our results show that somatic embryogenesis can be used as an alternative method to conventional propagation methods for rapid multiplication of uniform planting material in polyembryonic mango cultivars.

Inorganic Compounds that Aid in Obtaining Somatic Embryos.

Somatic embryogenesis (SE) is a process that allows formation of embryos from somatic cells; this biological process has different stages that first require micropropagation and conditioning of explant, and then induction, multiplication, development, and germination of somatic embryos (SoE), to obtain seedlings that will be acclimatized and grown in a greenhouse to further be cultivated in the field. Inorganic compounds are supplemented by macro- and micronutrients that can conform different culture media, and with other compounds such as a carbon source, vitamins, and plant growth regulators (PGRs), will direct the fate of the plant cells to obtain SoE that will regenerate into plants. The concentration of these inorganic compounds must be optimized, since at very high concentrations they can cause toxicity and at low concentrations they may not induce the desired response. The objective of this chapter is to describe the most significant advances in the use of inorganic elements during the different stages of SE, starting with the description of the most used basal media and later describing the use of the main studied mineral elements during establishment of SE.

Optimization of Somatic Embryo Regeneration of <i>Larix olgensis</i> Through Multiple Media

To establish a stable and efficient  Larix olgensis  somatic embryo regeneration system. In this study, immature zygotic embryos of  Larix olgensis  were used as explant materials for embryogenic callus induction, and embryogenic callus with good growth were selected to induce somatic embryo formation by liquid medium A, semi-solid medium B, and solid medium C. The callus induction medium is S medium+2,4-D 1.0 mg/L + 6-BA 0.5 mg/L + KT 0.3 mg/L, sucrose 20 g/L, agar 3 g/L; the subculture medium is S medium+2, 4-D 0.15 mg/L + 6-BA 0.05 mg/L + KT 0.05 mg/L, sucrose 30 g/L, agar 6 g/L. Mature medium A is S medium + PEG4000 100 g/L + ABA 15 mg/L + AgNO 3  10 mg/L, sucrose 45 g/L liquid medium. Mature medium B and C are mature medium A supplemented with 2 g/L and 10 g/L agar, respectively. The maturation time of this method is about 32 days, the synchronization rate is 78.0%, and the proportion of somatic embryos in normal morphology is 83.0%. Somatic embryo formation efficiency in normal morphology can reach 223.0 embryos/g embryogenic callus, somatic embryo germination rate can reach 47.2%. Compared with a single maturation medium, the maturation time is shortened, somatic embryo generation is more synchronized, and high somatic embryo yield and quality are improved. It provides a way for the commercialization of somatic embryo regeneration of  Larix olgensis , brings great convenience to large-scale breeding and reproduction, physiological and biochemical research, and genetic modification, and has great practical application value.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets.

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL–1 of L-glutamine, 1,050 mgL–1 of L-asparagine, 350 mgL–1 of L-arginine, and 35 mgL–1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.

Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

Plant regeneration via somatic embryogenesis in diploid cultivated cotton (Gossypium arboreum L.)

The diploid cotton species G. arboreum offers a better opportunity to elucidate gene structure and function as opposed to the allotetraploid cotton species. As a prerequisite for this, a reliable and efficient method of high frequency plant regeneration in G. arboreum must be established. In this study, callus was induced from hypocotyl, root and cotyledon of G. arboreum seedlings on MSB medium (MS salts and B5 vitamins) with 0.09 µM 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 2.32 µM KT (kinetin). During propagation, callus was effectively selected for subculture on different media based on cell morphology to induce embryogenic callus and somatic embryos. Embryogenic callus was induced on MS5 medium (MSB, 37.59 mM KNO3, 62.47 µM NH4NO3, 3% (w/v) glucose, 6.8 mM glutamine, 3.8 mM asparagine) from suspended cultures after several cycles of alternate solid MS3 medium—liquid MS4 medium culture, a key step in somatic embryogenesis. Solid MS3 medium was supplemented with 2.46 µM IBA (īndole-3-butyric acid), 0.93 µM KT, 6.8 mM Gln, 3.8 mM Asn; liquid MS4 medium was supplemented with 0.1 g/L NaCl, 0.1 g/L KCl and 0.1 g/L CuSO4. The solid–liquid alternate culture was effective for embryogenic callus induced in G. arboreum. MS5 medium with maltose (3% w/v) or glucose (1.5% w/v) + maltose (1.5% w/v) performed better than that added single glucose (3% w/v) for somatic embryo maturation and germination. The regenerated plants with well-developed roots were directly transferred to the soil or grafted onto germinated cotton plants. Plant regeneration via somatic embryogenesis in diploid cultivated species was established, but there is a need for improvement and optimization for gene functional analysis and gene editing in the diploid cotton species. Regenerated plants of G. arboreum, a cultivated diploid cotton, were obtained via somatic embryogenesis by adjusting the culture mode and medium compositions.

Plant Regeneration via Somatic Embryogenesis in Larix principis-rupprechtii Mayr

Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr) is a native conifer in North China with great economic and ecological values. Somatic embryogenesis (SE) is a powerful tool for the mass clonal propagation in plants. In this study, we described a high-efficiency SE system via indirect pathways and investigated the effect of genotype, culture conditions and phytohormones on SE. Immature zygotic embryos (IZEs) of L. principis-rupprechtii Mayr were used as explant materials. In the induction stage, embryogenic tissues (ETs) were induced on mLV medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 6-benzylaminopurine (6-BA). The initiation frequencies showed significant differences (p < 0.05) among 20 genotypes of open-pollinated mother trees with the highest induction frequency reaching 30%. For tissue proliferation, proliferation in liquid medium was more efficient compared with proliferation in semi-solid medium, providing a multiplication rate of 3.12 in an 8-day subculture period. As a necessary exogenous plant growth regulator (PGR) for somatic embryo maturation in conifers, abscisic acid (ABA) was optimized at 16 mg L−1 in this system. Next, an orthogonal test on osmotic pressure factors showed 50 g L−1 sucrose, 7 g L−1 phytagel and 75 g L−1 polyethylene glycol (PEG) was the optimal combination for somatic embryo maturation in L. principis-rupprechtii Mayr. Moreover, the dispersion culture method provided a more efficient somatic embryo maturation, up to 545 per gram of fresh weight (FW). Finally, 2 g L−1 of active charcoal (AC) was found to increase the somatic embryo germination rate to 63.46%. The improved protocol of SE will serve as a foundation for establishing mass propagation and genetic transformation of L. principis-rupprechtii Mayr.

Pengaruh Pemberian GA3 terhadap Perkecambahan Embrio Somatik Kakao

<p><em>Germination process of somatic embryos is one of critical steps in somatic embryogenesis. The conversion of somatic embryos into plantlets is still low and adding kinetin, ABA, NAA, and GA<sub>3</sub> to the germination medium of cacao somatic embryos may increase it. The study aimed to investigate the effect of GA<sub>3</sub> on somatic embryo germination at the cotyledonary phase. The study was conducted in the Indonesian Industrial and Beverage Crops Research Institute (IIBCRI) Tissue Culture Laboratory from May to November 2017. The study used a factorial completely randomized design (CRD) with seven replications. The first factor was the cacao genotypes, namely SCA 6 and MCC 02, and the second factor was four concentrations of GA<sub>3</sub>, namely 0.0; 1.0; 3.0; and 5.0 mg/l. Characters observed were the percentage of shooting cotyledonary embryos, the percentage of rooting cotyledonary embryos, the percentage of shooting and rooting cotyledonary embryos, percentage of browning cotyledonary embryos, root length, shoot length, and hypocotyl length at one and two months old. The results showed no interaction between genotypes and GA<sub>3</sub> concentrations on all observed characters. Adding GA<sub>3</sub> at 1,0 mg/l concentration tends to increase the percentage of shooting cotyledonary embryos of SCA 6 and rooting cotyledonary embryos of MCC 02. SCA 6 clone has a higher percentage of shooting cotyledone, shooting and rooting cotyledone, and browning cotyledone compared to those of MCC 02.</em></p>

Indirect somatic embryogenesis and genetic homogeneity assessment in Plectranthus bourneae Gamble an endemic plant to Western Ghats of Tamil Nadu, India

An effective somatic embryogenesis protocol was established for <i>Plectranthus bourneae</i> an Endemic plant to Western Ghats of Tamil Nadu, India. High frequency (77.28 %) of embryogenic callus was achieved from leaf explants on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D). The embryogenic callus was subcultured in a combination of 0.5 mg/L 2,4-D and 50 mg/L glutamine (GLU) which produced somatic embryos(84.22 %). Different stages of embryos (globular, heart, torpedo and cotyledonary stage) were observed. Maturation and the frequency of somatic embryo germination increased when transferred onto half-strength MS medium containing 0.5 mg/L gibberellic acid (GA3) and 0.5 mg/L 6-benzyladenine (BA). Plantlets were acclimatized successfully with 90 % survival. Histological studies revealed the development of primary and secondary embryos. The genetic fidelity of mother plants and in vitro raised plants was established by inter-simple sequence repeat (ISSR) markers. The established protocol assists the rapid production of true-to-type plants by somatic embryogenesis and hence could provide a valuable target material for genetic transformation experiments.

Indirect somatic embryogenesis and genetic homogeneity assessment in Plectranthus bourneae Gamble an endemic plant to Western Ghats of Tamil Nadu, India

An effective somatic embryogenesis protocol was established for <i>Plectranthus bourneae</i> an Endemic plant to Western Ghats of Tamil Nadu, India. High frequency (77.28 %) of embryogenic callus was achieved from leaf explants on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D). The embryogenic callus was subcultured in a combination of 0.5 mg/L 2,4-D and 50 mg/L glutamine (GLU) which produced somatic embryos(84.22 %). Different stages of embryos (globular, heart, torpedo and cotyledonary stage) were observed. Maturation and the frequency of somatic embryo germination increased when transferred onto half-strength MS medium containing 0.5 mg/L gibberellic acid (GA3) and 0.5 mg/L 6-benzyladenine (BA). Plantlets were acclimatized successfully with 90 % survival. Histological studies revealed the development of primary and secondary embryos. The genetic fidelity of mother plants and in vitro raised plants was established by inter-simple sequence repeat (ISSR) markers. The established protocol assists the rapid production of true-to-type plants by somatic embryogenesis and hence could provide a valuable target material for genetic transformation experiments.

Embriogénesis y organogénesis de aguacate criollo mexicano

La variedad Drymifolia de aguacate (Persea americana Mill.) es usada generalmente como portainjerto por lo que es importante implementar las técnicas de embriogénesis somática y la organogénesis in vitro para su mejora genética, conservación y propagación clonal. En este trabajo se evaluaron embriogénesis somática directa, indirecta y organogénesis de la variedad Drymifolia. La germinación de los embriones somáticos maduros se indujo con 0.5 mg L-1 de 6-N-bencil amino purina (BAP) y 1 mg L-1 de ácido giberélico (GA3). De las seis accesiones evaluadas, Celaya 79, Comonfort 53, San Miguel, BG24, BG181 y Zutano, solo San Miguel respondió al proceso de embriogénesis. En la embriogénesis directa la eficiencia más alta de regeneración se obtuvo con 0.2 mg L-1 de picloram (46%) y 10 mg L-1 de ANA (40%). En la embriogénesis indirecta la accesión San Miguel formó callos con 0.2 mg L-1 de picloram y tuvo una eficiencia de regeneración de 45% conservando su potencial de regeneración hasta por seis meses. Con respecto a la organogénesis, se cultivaron embriones cigóticos inmaduros decapitados en medio con reguladores de crecimiento o sin ellos y las seis accesiones respondieron positivamente a ambas condiciones. La accesión Comonfort 53 tuvo mayor eficiencia de regeneración (54%) con reguladores de crecimiento. Este estudio realizado del 2016 a 2018, proporciona un enfoque nuevo y prometedor para la regeneración y multiplicación de plantas de P. americana var. Drymifolia a través de embriones cigóticos.

Propagasi in vitro tanaman kurma (Phoenix dactylifera L.) pada bioreaktor dengan perendaman sesaat

The cultivation of date palm in Indonesia has increased since the last decade. However, the superior date palm seedlings are still limited and most of them are imported from other countries. The mass supply of superior date palm seedlings can be provided by in vitro propagation in the bioreactor. Therefore, the research was conducted to develop a protocol of date palm in vitro propagation by using Temporary Immersion Bioreactor (TIB). The in vitro propagation was carried out through somatic embryogenesis technique using meristematic tissues isolated from offshoots of date palm female clone cv. Zambli as explants. The explants were sterilized and then cultured to produce embryogenic calli and somatic embryos. Afterwards, somatic embryos germination and plantlets formation were conducted in TIB with treatments of immersion period: 3, 10, and 30 minutes every 6 hours, with 8 replications, The results showed that the optimal somatic embryo germination in TIB was with the immersion period of 30 min every 6 h, resulting in the most formation of shoots and fresh biomass weight increment up to nearly threefold in 6 weeks. Thereafter, plantlets formation in TIB with immersion period of 10 min and 30 min every 6 h exhibited similar performances in producing more plantlets with higher total fresh weight and better vigor than those of 3 min every 6 h. However, there were more rooted plantlets in the TIB with immersion period of 10 min every 6 h. Based on the results, an in vitro propagation protocol via somatic embryogenesis in TIB has been successfully developed for mass propagation of date palm cv. Zambli, which produced plantlets with good vigor and rooting.

In vitro conservation strategies for the critically endangered Malabar river lily (Crinum malabaricum Lekhak & Yadav) using somatic embryogenesis and synthetic seed production

Crinum malabaricum Lekhak & Yadav (Malabar river lily of the family Amaryllidaceae) is a critically endangered plant species and a natural source of galanthamine. Synthetic seeds production via somatic embryogenesis was adopted as in vitro conservation strategy and proliferation of Malabar river lily in this study. Murashige and Skoog's (MS) basal medium with 8.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and additives induced maximum percentage of somatic embryos (98.4%) at various stages with an average of 53.8 somatic embryos per inoculum/tri-scale explant. Histo-anatomical examinations showed that the meristematic cell layers of reduced stem (present between the scales) had the highest embryogenic capacity. The meristematic activity of the globular somatic embryos (SEs) assisted in further differentiation of distinct stages of embryogenesis. Synthetic seeds encapsulated with 2.0% sodium alginate and 100 mM calcium chloride resulted in the development of delicate beads with 92.5% post thawing germination potential after one year cold storage in dark at - 4 °C. The effect of 6-bezylaminopurine (BAP) and indole-3 acetic acid (IAA) on germination of SEs, plantlet retrieval, and shoot and root regeneration was significant as compared with combination of BAP and α-Naphthalene acetic acid (NAA). Highest 12.8 shoots per synthetic seed and 5.6 roots/ shoot were regenerated from encapsulated somatic embryos on MS medium + 2.0 mg L−1 BAP + 0.5 mg L−1 IAA + additives. This report could help in mass propagation of C. malabaricum, and could be applied for enhanced production of galanthamine using elicitors and through genetic transformations.

Somatic embryogenesis and plant regeneration in Piper aduncum L

An efficient protocol is reported for in vitro plant regeneration through somatic embryogenesis in Piper aduncum, a Brazilian Amazon species with high economic potential. The species is important due to a variety of components found in its essential oil, with emphasis on dillapiole. Leaf explants from five accessions identified for high oil yield and levels of dillapiole were evaluated for their embryogenic potential. To induce embryogenic calli, the explants were cultivated in MS medium supplemented with 5 mg L−1 of 1-naphthaleneacetic acid (NAA) and 2.5 mg L−1 of N6-benzylaminopurine (BAP) for 80 d. For somatic embryogenesis, the embryogenic calli were transferred to MS medium with 10 mg L−1 of NAA and 2.5 mg L1 of BAP and incubated for 45 d. The obtained somatic embryos were germinated in MS medium without regulators by 45 d and the obtained plantlets were subjected to acclimatization. Somatic embryos and calli from this process were subjected to anatomical and histochemical analyses. Biochemical analyses (total soluble sugars, starch, total amino acids, and proteins) were also performed to identify markers for embryogenic competence acquisition. In addition, the germination of somatic embryos was evaluated in a semi-solid and liquid system (R.I.T.A.® temporary immersion bioreactors). The obtained plants were evaluated for genetic fidelity using ISSR markers. The present study indicate that the accessions did not differ in embryogenic potential, with a mean percentage of calli with somatic embryos of 82.4%. Anatomical analyses confirmed the occurrence of the embryogenic route and the histochemical analyses identified starch grains in somatic embryos at different developmental stages. The biochemical analyses showed high total soluble sugars and total amino acids in embryogenic calli, marks of the acquisition of the embryogenic competence of P. aducum. The R.I.T.A.® temporary immersion bioreactors were highly efficient in the regeneration of somatic plants, with 100% germination. The plants regenerated in the semi-solid and liquid systems showed high genetic homogeneity. The survival rate of the acclimatized plants was 100%.

An advanced protocol for the establishment of plantlets originating from somatic embryos in Pinus massoniana.

This study describes critical factors affecting germination of somatic embryos and plantlet regeneration in Pinus massoniana. Somatic embryos from the same embryogenic line 27 of P. massoniana were used as test materials. The supplementation of activated charcoal (AC) in the medium was essential for the germination of mature somatic embryos, while the addition of excessive AC to the medium was prohibitive for somatic embryo germination. The highest germination rate was found on the medium containing 10g/l AC, and the addition of 5g/l AC to the medium was optimal to the growth of germinating somatic embryos. Thidiazuron (TDZ) was linearly related to the number of sprouting axillary buds. However, the growth of sprouting buds was retarded when > 4µmol/l TDZ was added into culture medium. Exogenous plant growth regulators added to the medium significantly improved the root regeneration capacity of shoots. The highest root regeneration rate was observed under the treatment of 1.2µmol/l ɑ-naphthaleneaceticacid (NAA) plus 2µmol/l paclobutrazol (PBZ), reaching 96.3%. One year after the field transfer, the growth performance of plant height, caliper, and survival rate for rooted shoots was significantly better than that of plantlets directly developed via somatic embryogenesis. The presented results provide useful instruction for the establishment of plantlets originating from somatic embryos, and would be able to make a great contribution to the clonal forestry of P. massoniana.

High-efficiency somatic embryogenesis techniques for different hybrids of cut lilies

Somatic embryogenesis (SE) is a unique method of in vitro regeneration for plant reproduction, germplasm resource preservation, and molecular-assisted breeding. Although lily is an internationally valued ornamental plant, its SE technology is still incomplete. Here, the effects of different explants, picloram (PIC) concentrations, and light conditions on somatic embryo induction, development, germination and seedling formation were studied in five Lilium varieties belonging to different hybrids. The embryo histomorphology, development and origins were observed, and a broadly efficient embryogenesis system was firstly established in Lilium. With leaf explants, all varieties tested underwent indirect somatic embryogenesis (SE). With scale explants, the Lilium Asiatic ‘Strawberry and Cream’, Lilium Oriental ‘Double Surprise’, and Lilium longiflorum Thunb. ‘White Heaven’ underwent indirect SE and the Lilium longiflorum × Asiatic hybrid ‘Eyeliner’ and Lilium longiflorum × Oriental ‘Triumphator’ underwent direct SE. Somatic embryogenesis in the above varieties involved four stages: globular, heart-shaped, torpedo-shaped, and cotyledon embryos. Two original SE pathways (endogenous and exogenous) were observed, and their appearance times differed. In indirect SE, exogenous embryos appeared first, followed by endogenous embryos; whereas in direct SE, the order was reversed. The different somatic embryo origins and the proportion of internal and external origins were closely related to the occurrence of somatic embryos by direct or indirect pathways and significantly affected the induction cycle of somatic embryos. Dark culture promoted lily somatic embryo induction, while 16 h/day light culture promoted germination, and the germination rates of all five lily varieties exceeded 90%. Murashige Skoog (MS) medium + (1.5–2.5 mg L−1) PIC + 0.2 mg L−1 1-Naphthaleneacetic acid (NAA) was suitable for SE from lily leaves and scales with an induction efficiency of 88–96%. Our research developed a widely applicable regeneration procedure for different hybrids of lily via somatic embryogenesis. Morphohistological analysis confirmed the origins, developmental stages and dynamic changes of cells during in vitro morphogenesis. This study will provide a valuable reference for the propagation of Lilium.

Induction of somatic embryogenesis in different explants from Ormosia henryi Prain

An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain, which is an important precious tree in the timber industry. Five types of explants, six types of basic media and different plant growth regulators (PGRs) were used for embryogenic callus (EC) initiation, EC proliferation, somatic embryo (SE) induction and SE germination. The EC and SE induction rates from mature zygotic embryos (MZE) were higher than those from stem segments, cotyledons, leaves and hypocotyls. MZE were used as explants, and the EC induction rate was greater than 30% on B5 medium supplemented with 1.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l naphthaleneacetic acid (NAA) or 0.2 mg/l BA and 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The highest induction rate (22.83%) for SE was obtained with B5 medium supplemented with 0.5 mg/l kinetin (KT) and 1.0 mg/l 2,4-D. The EC proliferation rate and SE number in the suspension culture were increased compared to those in the solid culture. Fifty-four percent of SE developed to the cotyledonary stage on B5 medium supplemented with 0.5 mg/l BA and 0.2 mg/l NAA. The number of cotyledon embryos was the highest on medium supplemented with 0.5 mg/l thidiazuron (TDZ) and 0.2 mg/l NAA. Histological analysis confirmed the bipolar organization of the somatic embryos, which were comprised of shoot and root meristems, and showed that SE was characterized by globular, heart-shaped, torpedo-shaped and cotyledon stages in O. henryi, the developmental process of which was similar to that of zygotic embryos. An efficient in vitro plant regeneration method via somatic embryogenesis has been established in Ormosia henryi Prain using mature zygotic embryos as explant.

Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths.

Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or β-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5g/L gelrite, and 0.2mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.

Use of Plant Antimicrobial Peptides in in vitro Embryogenic Cultures of Larix sibirica

The effect of plant antimicrobial peptides on the initiation of callus and embryonal suspensor masses, formation of somatic embryos, and germination of regenerants of Siberian larch has been studied. Protein/peptide extracts isolated from Amarantus retroflexus (seeds), Nigella sativa (seeds), and Elytrigia elongata (spikelets) have been used as objects of plant origin. Peptides have been introduced into the nutrient media at the stage of initiation of embryogenic cultures and somatic embryo germination. The stimulating effect of peptides on the formation of embryogenic cultures of Siberian larch has been found. No other differences in the dynamics of growth in the control and experimental regenerants have been observed. This study is supposed to contribute to enhancing the immunity of the clonal planting stock of Siberian larch.

Differential Influence of Growth Regulators during Somatic Embryogenesis of Gynodioecious Papaya Varieties‘CO.7’ and ‘Red Lady

The study involved two auxins viz., 2,4-D (2,4-Diclorophenoxyacetic acid) and picloram at three different concentrations (1,2, 3 mg/L) in full strength MS media to study their comparative influence on induction of somatic embryogenesis from immature zygotic embryos of two gynodioecious varieties of papaya ‘CO.7’ and ‘Red Lady’. In papaya cultivar ‘CO.7’, 2,4-D at 2 mg/L gave the highest callus induction frequency of 90.93%, whereas comparatively higher concentration of 3 mg/L 2,4-D was found suitable for ‘Red Lady’ (87.26%). Although there was profuse callus formation, 2 mg/L 2,4-D recorded comparatively higher frequency of embryogenic calli in ‘Red Lady’ (51.67%) when compared to ‘CO.7’ (30.00%). Somatic embryo maturation was achieved upon transfer of embryogenic calli exhibiting globular stage embryos on to maturation medium (MS medium + ABA (Abscisic acid) and BAP (Benzyl amino purine) in different concentrations + glutamine 400 mg/L). In the maturation medium, the combination of 1.5 mg/L ABA and 0.4 mg/L BAP registered better conversion of the globular embryo to cotyledonary embryos than other levels. The frequency of somatic embryo germination was higher in ‘Red Lady’ (50.00%) as compared to ‘CO.7’ (31.67%) on half-strength MS medium devoid of growth regulators.