Histology and Morphology of Asparagus Somatic Embryos

Plant Cell Reports, 1991

The explant used to initiate embryogenic calIus and the growth regulators used in subsequent induction (IM) and embryo development media (EDM) both influenced rate of somatic embryo development and conversion to plantlets in asparagus. Embryogenic callus derived from spear-cross sections (SS), in vitro crowns (IVC) and lateral buds (LB) was cultured on IM of MS salts and vitamins with 2,4-D or NAA at 0, 0.01, 0.1, 1.0 or 10 mg/l and kinetin at 0, 0.1, 1.0 or 10 rag/1. The auxin 2,4-D at 1-10 mg/t, in combination with kinetin at 0-1 rag/l, in IM induced the highest frequency of embryos after four weeks; callus derived from SS, IVC and LB had means of 394, 382, and 344 small globular embryos, and 4, 11 and 9 bipolar embryos per gram of callus, respectively. After 6 weeks on EDM, 128, 116 and 51 bipolar embryos (4-7 mm in length) occurred per gram callus and 4.5, 1.4 and 2.1 embryos converted for IVC, SS and LB, respectively. NAA at 1-10 rag/I, in combinations with kinetin 0-1 rag/t, yielded means of 64, 175 and 225 small globular embryos per gram callus on IM for SS, IVC and LB, respectively. NAA promoted a higher rate of embryo development: means of 27, 54 and 91 bipolar embryos per gram callus for SS, LB and ]VC, respectively, on EDM. There were 0.5, 9.4 and 11.9 plantlets from these respective callus sources. There was no difference between ldnetin levels of 0-1 mg/l on callus growth and embryogenesis, whereas, 10 mg/l in IM was inhibitory. ABBREVIATIONS 2,4-D-2,4-dichlorophenoxyacetie acid, EDMembryo development medium, LAA-indole-3-aeetie acid, IMinduction media, IVC-in vitro crowns, LB-lateral bud, LS-Linsmaier and Skoog (1965), MS-Murashige and Skoog (1962), NAA-naphtha[eneacetie acid, SS-spear-cross section Materials and methods Spear segments ($8), lateral buds (LB), and in vitro maintained crowns

Journal of Plant Biochemistry and Biotechnology, 2007

An efficient protocol has been developed for plant regeneration in Asparagus densiflorus (Kunth) Jessop cv Sprengeri (Asparagaceae) through somatic embryogenesis from spear sections. Callus culture was initiated on Murashige and Skoog (MS) medium formulation containing α-naphthaleneacetic acid (5.37 μM), indole-3-acetic acid (5.71 μM) and 6benzylaminopurine (2.22 μM). The callus became embryogenic by transferring to 2, 4-dichlorophenoxyacetic acid (4.52 μM) containing MS medium. Somatic embryos developed and matured vigorously on MS medium with NAA (1.07 μM), 6-γ-γdimethylaminopurine (9.84 μM) and abscisic acid (1.89 μM). Mature bipolar embryos were converted efficiently into plants on MS medium in the presence of low level of kinetin (2.32 μM). Regenerated plants showed 80% survival after transfer to field. These plants were all diploid (2n=60). Peroxidase activity was maximum in the embryogenic callus as documented from the gel as well as spectrophotometric analysis.

CYTOLOGIA, 2003

A tetraploid callus line of Asparagus officinalis L. was identified from 4 regenerated callus lines. Plants were regenerated from these callus lines following somatic embryogenesis and the tetraploid clone of this species was established in the field with 80% survival rate. The embryogenic callus was induced in Murashige and Skoog's (MS) medium in presence of a-naphthaleneacetic acid (NAA) (0.2 mg/l) and kinetin (0.02 mg/l) and proliferated as well as maintained in 2,4dichlorophenoxyacetic acid (2,4-D) (1.0 mg/l) containing MS medium. Somatic embryos were initiated in Gelrite-solidified MS medium with variable levels of NAA and 6-(g-g-dimethylallylamino) purine (2ip). A higher level of carbohydrate enhanced embryo conversion efficiency. The embryos induced in presence of 10% glucose for 2 weeks and subsequently transferred to 2% sucrose level showed higher conversion rate than those maintained in 3% sucrose concentration. Karyotype analysis of diploid and tetraploid clones revealed exact duplication of the diploid set in tetraploid plants.

ABSTRACT Somatic embryogenesis and plantlet formation were obtained from callus derived from the subapical region of spears of Aspara$us cooperi Baker. Callus was obtained in Murashige and Skoog's medium supplemented with l-naphthaleneacetic acid and kinetin. Increase in the concentration of potassium nitrate in subsequent subcultures resulted in the formation of embryos. Rapid multiplication of embryos was secured on transfer to a medium containing a different source of nitrogen and a low level (0.01 rag/l) of gibberellic acid. Media containing zeatin or gibberellic acid led to the formation of complete plantlets from embryos. Regenerated plants were cytologically and phenotypically stable.

Plant Biotechnology, 1998

Plant Science, 1999

We study the induction of somatic embryogenesis using an original Asparagus model: a mutant type which an important somatic embryogenesis and its non-embryogenic wild type used as control. We describe the histological events and the modifications of protein patterns occurring during the initiation of somatic embryogenesis in cultures of apices. Using the mutant type apices, pro-embryogenic masses and embryogenic cells are observed at the periphery of the callus after transfer on hormone-free medium while in the same culture conditions, the wild type calli never show embryogenic structure. Analysis of the tissular proteins during the culture leads us to classify 116 proteins into 20 groups potentially related to somatic embryogenesis. Two of these groups are particularly interesting as they correspond to polypeptides that are only related to one of the two types: six polypeptides (group 1) are specific to the mutant type apices and could be related to the competence of the mutant tissues with respect to somatic embryogenesis. Eleven proteins (group 7) are detected specifically in the wild type tissues and their presence could be related to an inhibition of the somatic embryogenesis ability.

Genetics and Molecular Biology, 2005

Somaclonal variation in plants regenerated by organogenesis from long-term cultured calluses of two diploid staminate genotypes of Asparagus officinalis cv. Argenteuil was characterized by plant phenotype, ploidy, meiotic behavior, pollen viability, fruit and seed set, and AFLP profiles. Phenotypic deviations from the donors were detected in foliage color, flower size, and cladode and flower morphology. Ploidy changes were observed in 37.8% of the 37 regenerants studied. Meiotic alterations in 12 out of 21 regenerants included laggards, dicentric bridges, micronuclei, restitution nuclei and polyads. Of the 408 AFLP markers screened in 43 regenerants and the donors, 2.94% showed polymorphism. High pollen viability was observed in the 22 regenerants analyzed. All crosses between one pistillate plant and 35 regenerants, as well as the controls, produced fruits and seeds; however, no plump seeds resulted in 35.3% of the crosses with regenerants, and no seeds germinated in 12.5% of those with apparently normal seeds. Fruit and seed set was similar in crosses with diploid regenerants with normal meiosis and the controls but was lower in crosses with diploid and polyploid regenerants with abnormal meiosis. Our results show that the regenerated plants exhibited conspicuous somaclonal variation that could be eventually exploited for in vitro selection systems.

In Vitro Cellular & Developmental Biology - Plant, 2006

To pursue the causal factors of ploidy variation in tissue culture of asparagus (Asparagus officinalis L.), ploidy status of stem explants and the calluses induced from these explants were examined using flow cytometry (FCM). The frequency of higher ploidy (.8C) cells in the stem explants increased with increasing distance from the shoot apex. Calluses derived from sections far from the apex also showed higher ploidy distributions, and also higher ploidy (16C) in general than was observed in the explant tissue. Detailed studies using FCM and charge-coupled device (CCD)-equipped fluorescence microscopy showed that 16C cells appeared in the explant tissues within 1 wk of culture, possibly induced by the plant growth regulators (PGRs), especially 1-naphthaleneacetic acid (NAA), in the culture medium.

Journal of Scientific Research in Science, 2018

Cellular totipotency is one of the fundamental principles of plant biotechnology. The mature embryo is increasingly recorded as a valuable explant for somatic embryogenesis in rice biotechnology. In present study, rice cells dedifferentiation, proliferation and redifferentiation were investigated by culturing mature rice (Oryza sativa L. cv. Sakha 101) embryos in modified MS media fortified with different growth regulators alone and in combination. Mature embryo tissues competent for tissue culture and the chronological changes of cells morphology and histology were observed. The results showed that callus was induced only from mature rice seed (explant) cultured on MS media supplemented with 2, 4-D (2 or 2.5 mg/l) alone while the rest treatments showed negative response. Callus was initiated after 5 days of culture in MS media fortified with the lower 2, 4-D dose as clusters of undifferentiated cell masses while callus initiation was delayed 4 days more by increasing the applied dose. At morphological level, Pale yellowish and friable calli was noted in both tested doses. Calli texture exhibited different appearance, while was slightly nodular in the lower dose it was soft in the higher one. High callus induction frequency (70%) was estimated for 2mg/l 2, 4-D application while decreased frequency (40%) was concomitant with dose increment. Histological analysis for somatic embryogenesis revealed that within two weeks of culturing explants on callus induction medium (CIM), somatic embryos development began as clusters of embryonic cells at the peripheral parts of the proliferated calli while nonembryonic cells were observed at the inner regions of the induced callus. Embryogenic cells at the outer cell layer were observed as small and isodiametric with dense cytoplasm and clear nucleus located in the center of the cells, whereas the non embryogenic cells were large, vacuolated and had a very small nucleus located near the cell wall. Embryogenic cells undergo series of ordered divisions and protodermis observed surrounding globular embryo was recorded at the end of culturing in CIM fortified with lower 2, 4-D dose. On the other hand, culture on MS fortified with higher dose delayed rice cells differentiation and globular stage was recorded two days after subculture embryonic callus into free hormone MS medium. After 2 days of subculture into free hormone MS medium, heart shaped embryo was observed in low dose of 2, 4-D. This study assists to draw attention to the use of a histological approach as a helpful tool to follow the chronological series of embryo development in vitro.

Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Animal Science and Biotechnologies, 2007

There are various reasons to consider plant somati c embryogenesis as an useful tool in the researches of plant embryology: the similarity of z ygotic and somatic embryogenesis. the visibility of somatic embryos. the availability of a big amount of somati c embryos for biochemical analyses. The aim of this paper is to present a comparison between the zygotic and the somatic embryogenesis. to show a method for the investigation of the in vitro embryo maturation. an d to discuss the functionality of synchronous and s equential embryogenic systems.