影响香根草体细胞胚胎发生和植株再生因素初探

为了探索提高香根草(Vetiveriazizanioides)遗传种质改良效率的有效途径,初步研究了影响香根草体细胞胚胎发生和植株再生的若干因素。以MS培养基为基本培养基,附加不同配比的生长素和细胞分裂素,对香根草的腋芽及无菌不定芽进行离体培养。结果表明,2,4-D是诱导体细胞胚胎发生的关键因素,当培养基中只含2,4-D而不含或少含细胞分裂素(6-BA)时,外植体经由体细胞胚胎发生途径形成再生植株。愈伤组织诱导频率在有的品种之间差异显著,最高的达到96.7%(cv.Zomba),最低的不到30%(cv.Malaysia);而有的品种之间差异不显著(例如cv.Kandy和cv.Sunshine)。胚性愈伤组织的再生能力可以长期保持,从未经继代培养的到持续继代第23代的胚性愈伤组织,再生频率都在80%以上,而且再生植株的生长良好。在3-7℃的低温下进行分化培养时,仍然有40.5%-50.0%不同世代的胚性愈伤组织还保持着再生能力。

细胞分裂素类物质在植物体细胞胚发生中的作用

本文对细胞分裂素及其类似物在植物体细胞胚发生中的作用的研究历史、现状及前景进行了评述。

Histological Study on Soybean Somatic Embryogenesis

Soybean somatic cell could induce the development of embryoid which was similar to embryo morphologically and structurally. Somatic embryogenesis system of soybean was used to conduct genetic transformation of soybean because of its several advantages such as higher transformational efficiency, beetter synchronism and fewer plant chimeras among transgenic plants. After infected with agrobacterium tumefaciens,the initiation, differentiation and development of young cotyledon embryogenic cell of soybean which was cultured on selective culture medium with kanamycin were investigated through histological study. The result showed that somatic embryo was differentiated in non-bud differentiation way. The embryogenic cells were differentiated from epidermis of explant or cells in 1 layer or 2 layers, with the division of embryogenic cells and degradation and disorganization of surrounding cells, the embryogenic cells would form embryoid with analogous suspensor structure. Later, globular embryoid would extrude from epidermis then developed into heart-shape embryo. The experiment was expected to provide theoretical reference for the construction of high transformational system of using plant somatic embryogenesis induced by young cotyledon of soybean.

Studies on Parameters Affecting Embryogenesis of Protoplasts Isolated from Suspension of Embryogenic Cells in Loblolly Pine

Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.

Somatic embryogenesis and peroxidase activity of desiccation tol-erant mature somatic embryos of loblolly pine

White, translucent, glossy mucilaginous callus was initiated from the mature zygotic embryos explants on callus induction medium with 2,4-D, BA, and kinetin in the 3-9th week of culture. This type of callus induction occurred at a lower fre-quency with either a-naphthaleneacetic acid (NAA) or IBA (both 8 mg/L). White, translucent, glossy mucilaginous callus was embryogenic and mainly developed from the cotyledons of the mature zygotic embryo. Somatic embryos were formed on dif-ferentiation medium. Desiccation tolerance can be induced by culturing somatic embryos of loblolly pine (Pinus taeda L.) on medium supplemented with 50 mm abscisic acid (ABA) and/or 8.5% polyethylene glycol (PEG6000). Scanning electron micros-copy of desiccated somatic embryos showed that the size and external morphology of the desiccation tolerant somatic embryos recovered to the pre-desiccation state within 24-36 h, whereas the sensitive somatic embryos did not recover and remained shriveled, after the desiccated somatic embryos had been rehydrated. Peroxidase activity of desiccated somatic embryos in-creased sharply after 3 days of desiccation treatment, and desiccation tolerant somatic embryos had higher peroxidase activity compared to sensitive somatic embryos. Higher peroxidase activity of desiccation tolerant somatic embryos was possibly ad-vantage of catalyzing the reduction of H2O2 which was produced by drought stress, and protecting somatic embryos from oxida-tive damage.

Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis

Somatic cell nuclear transfer （SCNT） and parthenogenesis are alternative forms of reproduction and development, building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin, respectively. In the preceding paper （Sun F, et al., Cell Res 2007; 17：117-134.）, we showed that an ＂erase-and-rebuild＂ strategy is used in normal development to transform the maternal gene expression profile to a zygotic one. Here, we investigate if the same strategy also applies to SCNT and parthenogenesis. The relationship between chromatin and chromatin factors （CFs） during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein assays. Results from these studies indicated that soon after nuclear transfer, a majority of CFs dissociated from somatic nuclei and were redistributed to the cytoplasm of the egg. The erasure process in oogenesis is recaptured during the initial phase in SCNT. Most CFs entered pseudo-pronuclei shortly after their formation. In parthenogenesis, all parthenogenotes underwent normal oogenesis, and thus had removed most CFs from chromosomes before the initiation of development. The CFs were subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized embryos. Based on these data, we conclude that the ＂erase-and-rebuild＂ process observed in normal development also occurs in SCNT and in parthenogenesis, albeit in altered fashions. The process is responsible for transcription reprogramming in these procedures. The ＂erase＂ process in SCNT is compressed and the efficiency is compromised, which likely contribute to the developmental defects often observed in nuclear transfer （nt） embryos. Furthermore, results from this study indicated that the cytoplasm of an egg contains most, if not all, essential components for assembling the zygotic program and can assemble them onto appropriate diploid chromatin of distinct origins.

Conservation of Iris bismarckiana Regel （Iridaceae） Using Plant Regeneration via Somatic Embryogenesis

In an attempt to propagate and conserve the rare, showy bulbous plants of Iris bismarckiana, newly recorded to the flora of Jordan and to contribute to the conservation the wild lris species in Jordan, a simple rapid, time consuming protocol has been developed using plant regeneration via somatic embryogenesis. Somatic embryogenesis was induced in zygotic embryo culture on Murashige and Skoog （MS） medium supplemented with 2,4-dichlorophenoxy acetic acid （8 mg/L） as the sole plant growth regulator, where both embryogenesis calli and somatic embryos were induced. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos successfully from embryo clusters and transferred onto fresh MS medium. Data obtained were analyzed as a complete random design with three replications. Calli fragments that were transferred to embryogenesis induction medium （EIM） produced white embryo-like globular structures within two weeks. Within three more weeks, clusters of structures at various stages of development could be found on the same callus. The applied technique is rewarding and encouraging for further research on the endangered wild species of Iris in Jordan.

Characterization of Pro-embryogenic Calli and Somatic Embryogenesis of Byrsonima intermedia A. Juss.

Byrsonima intermedia A. Juss. is a species from the Brazilian Cerrado that produces edible fruits and, in common with other species from the Byrsonima genus, has pharmacological potential. Previous attempts to propagate the species through conventional methods showed difficulties. Thus, the purpose of this work was to characterize pro-embryogenic masses of Byrsonima intermedia callus, aiming for their in vitro propagation through somatic embryogenesis. Leaf segments from in vitro germinated seedlings were employed as explants for callus production. The calli were then subcultured and exposed to dyes to fulfill their embryogenic potential. Digitalizations of the cytological preparations were made in order to measure the area that was stained by both Aceto-Carmine and Evans-Blue, using image tool software. Somatic embryos were induced after treatments with l-naphthaleneacetic acid （NAA）. The percentages of double-colored areas （by Aceto-Carmine and Evans-Blue） were calculated and the data were analyzed by using the Skott-Knott test （P ≤ 0.05） and, the embryogenic callus, as well as the formation of somatic embryos were analyzed by using the Krsuskal-Wallis rank test （P ≤0.05）. The results show that double coloration is effective at identifying cells showing embryogenic potential. Early callus subculture phases show a larger percentage ofembryogenic area （83%） Somatic embryos were induced by using high auxin level.

In Vitro Picloram-lnduced Somatic Embryogenesis from Leaflets of Cherry （Prunus incisa Thunb,）

Cherry regeneration via somatic embryogenesis is a powerful tool to breeding. In this way, the embryogenic capacity of Prunus incisa specie has been tested from leaves under different interactions of picloram concentrations and darkness exposures. Induction culture was achieved on MS medium supplemented with picloram concentrations at 0.5, 1 and 1.5 mg.L1 and submitted to 10, 20, 30 and 40 days of darkness. The best rate of embryogenic leaves was obtained with the interaction of 30 days darkness exposure＊ 1 mg.L^-1 picloram. According to their age, leaves were differently reacted to somatic embryogenesis; indeed, the 2nd expanded leaf from the apex was the most embryogenic one. Concerning the effect of additional auxin to picloram （1 mg·L^-1）, IAA at 0.1 mg·L^-1 and IBA at 0.1 mg·L^-1 gave significantly higher induction rates than all other concentrations, but regenerating somatic embryos showed some teratological abnormalities probably due to seconda;y embryogenesis. At the opposite, NAA at 0.5 mg·L^-1 didn＇t improve embryogenic rate but affected positively embryo development. Furthermore, embryogenesis preferentially took place on the basal part of leaf. Satisfactory rates of somatic embryogenesis are obtained but further improvement remains possible.

In Vitro Regeneration of Commercial Sugarcane （Saccharum spp.） Cultivars in Nigeria

The effect of different concentrations of 6-benzylaminopurine （BA） with or without 0.2 mg/L NAA on in vitro regeneration of sugarcane （Saccharum spp.） cultivars SP726180, B47419, M1176/77 and M2119/88 were evaluated. Leaf base explants were cultured on Murashige and Skoog （MS） basal medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid （2,4-D） for 4 weeks. Thereafter, induction of somatic embryogenesis was observed following the transfer of resulting calli to 2,4-D-free medium for another 4 weeks. Regeneration was achieved by transfer of the embryogenic calli to regeneration media fortified with different concentrations of BA ＋ tt-naphthylacetic acid （NAA）. The number, length and vigor of shoots produced in all the genotypes were highest on media supplemented with 1.0 and 1.5 mg/L BA with and without 0.2 mg/L NAA. Among the genotypes tested, B47419 and M1176/77 recorded highest number of shoots, while maximum shoot length and crop vigor was obtained with M1176/77. Induction of callus with 3.0 mg/L 2,4-D and its subsequent incubation on 2,4-D-free media, followed by regeneration on media supplemented with 1.0 or 1.5 mg/L BA with 0.2 mg/L NAA was found to be efficient for in vitro regeneration of the sugarcane genotypes used in this study. This protocol could be applied for micropropagation of other elite genotypes.

Effect of Abscisic Acid on NaCI Stressed Callus Proliferation and Plant Regeneration in Rice

In rice, 2,4-Dichlorphenoxyacetic acid （MS2） has been considered best for callus induction and combination ofNAA, IAA, ABA （MS3） for somatic embryogenesis. Efficient plant regeneration was observed when MS basal salts supplied with 3% sorbitol and 3% maltose as carbon sources without hormones （MSac）. During this experiment, effect of sodium chloride （NaCl） and abscisic acid （ABA） was assessed on callus growth, plant regeneration and root induction efficiency of rice （Oryza sativa L.） varieties IR-6 and Basmati-370. Callus proliferation rate was highly decreased in both varieties on MS2b （100 mol.m3 NaCl ＋ 0.5 mg＇L1 ABA） than MS2a （100 mol＇m3 NaCl） cultures significantly. Proline, glycine-betaine and reducing sugars were increased significantly in MS2a and MS2b callusing cultures. However, total proteins were decreased in MS2a, while slightly increased in MS2b. Maximum plant regeneration （9.42 ±0.54 and 10.67 ±0.50 plantlets.callus1） from somatic embryos was observed on MS4c in IR-6 and Basmati-370, while 1.56 ± 0.06 （IR-6） and 0.95 ±0.05 （Basmati-370） plantlets callus1 were observed on MSab （100 mol·m3 NaCl）. No plant regeneration was observed on MS4b （100 mol·m3 NaCl ＋ 0.5 mg·L-1 ABA） medium in both varieties. Inhibition of root induction efficiency was high in MSsb （100 mol·m3 NaCl ＋ 0.5 mg·L-1 ABA） than MS5a （100 mol·m3 NaCl） in the stressed cultures （P 〈 0.05）. In this experiment, it was concluded that ABA involved in somatic embryogenesis and elevation of NaCl stress, while it causes inhibition of cell＇s growth as well as its regeneration into plantlets from somatic embryos.

Preliminary Investigation on Somatic Embryogenesis from Immature Cotyledon Explants of Shea （Vitellaria paradoxa G,）

Long juvenile phase and lack of effective protocols for large scale vegetative propagation are limitations to domestication and improvement of the shea tree. The present study seeks to develop a protocol for plant regeneration of shea （Vitellaria paradoxa） from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium （MS） containing 3% sucrose, 0.24% Phytagel, and various concentrations of 2,4-Dichlorophenoxyacetic acid （2,4-D） after four weeks of culture in darkness. Rates of embryogenic callus induction were significantly affected by the addition of 2, 4-D to the medium. Within 28 days of culture, the highest percentage of embyogenic calli （77.61%） occurred on MS media containing 0.45 ~tM of 2,4-D in the dark. Somatic embryos were obtained by culturing embryogenic callus （in the dark） on MS medium fortified with 3% sucrose, 0.24% phytagel and devoid of growth regulators. Culturing at 16 h photoperiod restricted both the induction of embryogenic calli cultures and somatic embryos. Somatic embryos germinated, developed shoots and rooted vigorously on MS medium devoid of growth regulators. Germinated plantlets were acclimatized, successfully.

A Review of Somatic Embryogenesis in Cucurbitaceae

Somatic embryo is widely used in genetic engineering and cell engineering. This paper reviewed the recent research results of somatic embryogenesis in Cucurbitaceae plants. Somatic embryogenesis is controlled by many factors such as genotype, explant type, seedling age, basal culture medium, carbohydrate, nitrogen, growth regulators, additives and illumination et al.. Abnormality, desynchrony and browning are the main problem existing in Cucurbitaceae somatic embryogenesis. Then some ideas on how to obtain high quality somatic embryo are given. At last, we forecast the application of somatic embryo in breeding of Cucurbitaceae plants.

Media Appraisal for Somatic Embryogenesis of Elite Inbred Lines of Maize

Several protocols have been published for somatic embryogenesis in maize. It is essential to compare different protocols on selected germplasm at initial stages of tissue culture to identify a protocol that would yield optimum results. The ultimate goal is to use in vitro system for selecting and generating stress tolerant maize germplasm. Immature embryos of the elite inbred lines of maize （87014 ×Z28-11 and POP43SRS5-57） were cultured on three different semi-solid media （0.5% agar） and the effectiveness of the media for somatic embryogenesis assessed over a period two weeks of sub-culturing. A significant difference was observed among the media in proportion of potentially embryogenic calli and callus cluster size at 1 and 2 weeks after culturing. N6 medium supplemented with casaminoacid （100 mg/L）, 2, 4-dichlorophenoxy acetic acid （1 mg/L）, L-proline （25 mM） and sucrose （2%）, and Murashige and Skoog （MS） medium supplemented with 2, 4-D （2 mg/L） and sucrose （3%） gave higher proportion of potentially embryogenic calli and callus size than MS medium supplemented with casaminoacids （100 mg/L）, 2, 4-D （2 mg/L）, abscissic acid （3.3μM）, and silver nitrate （195 μM） and sucrose （3%）. The difference between genotypes is not significant

Dicamba Growth Regulator Promotes Genotype Independent Somatic Embryogenesis from Immature Zygotic Embryos of Tropical Maize Inbred Lines

Maize is one of the most important cereal crops in Sub-Saharan Africa and an important source of energy for humans. However, the difference in the dedifferentiation frequency of immature embryos among various genotypes indicates that callus induction and genetic transformation is dependent on the genotype. This phenomenon is an impediment in the fundamental process of improving tropical maize germplasm especially through genetic engineering. Here, five tropical maize （Zea mays L.） genotypes, CML 216, CML 144, A 04, E 04 and TL 21, were evaluated for callus induction on MS medium supplemented with the growth regulator dicamba. Embryogenic and non embryogenic callus induction was independent ofgenotype when young immature embryos, 12 days after pollination （DAP） were used for tissue culture in combination with dicamba. The optimal concentration of dicamba for induction ofembryogenic callus in all the genotypes was 3 mg/L, which was also the concentration at which non embryogenic callus formation was lowest. The frequency of embryogenic callus induction ranged from 35% to 79% among the five genotypes and somatic embryos regenerated R0 shoots that produced normal R1 progenies. This regeneration method is expected to facilitate the development of a more efficient genotype independent Agrobacterium- mediated transformation system for tropical inbred lines.

Effect of Different Hormone Combinations on Somatic Embryogenesis in Cotton Cultivar Xinluzao 33(Gossypium hirsutum L.)

[Objective] This study was conducted to evaluate the efficiency of somatic embryogenesis and plant regeneration for an upland cotton cultivar Xinluzao33 under the induction of different hormone combinations and thus to determine the optimal hormone combination. [Method] Calli of Xinluzao33(Gossypium hirsutum L.) were induced from seedling hypocotyl tissue by a range of DK and BK combinations. Embryogenic calli and embryos were induced on callus-inducing medium(CIM) without any hormones. Callus appearance and quality were compared to determine which medium was the optimal for callus induction. Embryogenesis ratio was calculated to determine which medium was the best for somatic embryogenesis and plant regeneration. [Result] Callus induction rate was 100% in all the 12 hormone combinations.The calli were yellow or kelly, and their texture was loose or soft under low concentrations of DK combinations, green or white, variably compact under high concentrations of DK combinations. The calli induced by BK combinations were kelly or green, covering creamy white substance. The best medium for callus induction was DK6(0.05 mg/L 2, 4-D and 0.10 mg/L KT). Embryogenic calli were successfully induced from all the combinations. The efficiency of embryogenic callus induction,embryogenesis, and plantlet regeneration were significantly different among the 12 combinations. The result showed that the embryogenesis ratio was the highest in BK3 combination(0.50 mg/L IBA and 0.50 mg/L KT), 72.86% of embryogenic calli differentiated into somatic embryos after being cultured on CIM for 80 d, and80.93% of the somatic embryos finally regenerated into plants on SEM(somatic embryo induction medium). [Conclusion] These results indicate that hormone combination BK3(0.50 mg/LIBA and 0.50 mg/L KT) was the best medium for somatic embryogenesis and plant regeneration from Xinluzao33.

Direct Differentiation of Plants from Small Cell Clusters of Indica Rice in Suspension Culture

The major factors affecting plant regeneration in suspension culture were investigated.The resultsshow that,in order for cell clusters to differentiate directly in liquid medium,it is essential toestablish an embryogenic callus line with high potential for plant regeneration.Embryogenic calliwere suspended in AA basic medium supplemented with 2,4-D 1 mg/L,6-benzylaminopurine 0.5mg/L,CH 300 mg/L,sucrose 3％ and mannitol 3％ and subcultured 7 days for each passage.Aftermore than 6 months of culturing,a fine suspension culture of small cell clusters(SCC)wasestablished.The SCC,80-270μin diameter,were then transferred to a liquid medium(MSsupplemented with NAA 0.01 mg/L and 4-pyridylurae(4-PU)0.5 mg/L)and allowed to grow instationary culture.Finally direct differentiation from SCC was observed.Several factors in suspension-subculture exerted strong after-effects on differentiation ofSCC.Basicmedia,kinds and concentration combinations of auxin and cytokinin in the subculture media,duration of shaking at each subculture passage,and amount of packed cell volume transferred to newmedium at each subculture passage and so on,all these affected the frequency of differentiation ofSCC.Higher concentrations of NAA and kinetin in the differentiation medium inhibited the directdifferentiation of SCC.Low concentrations(0.01-0.1 mg/L)of NAA with 4-PU in thedifferentiation medium were helpful for the direct differentiation of SCC.The differentiated clusterspossessed typical embryogenic structure from which normal plantlets could develop after transferringto agar medium.