Induction of Endosperm Calluses and Regeneration of Endosperm Plantlets of Asparagus officinalis

The endosperm callus has been induced from the young endosperm of Asparagus officinalis L. on the MS supplemented with auxin. The induction frequency of callus amounts to 65.9%–83.1%. When the callus was transferred to the medium supplemented with lower concentration of NAA 0.1 ppm or containing BA 1 ppm and NAA 0.5 ppm, the differentiation of shoots, roots and a few embryoids began to occur. A few calluses and embryoids can develop into plantlets. The chromosome number in the cells from the same root tip and shoot apex of endosperm plantlet is very unstable. They can be euploids (n=10, 2n=20, 3n=30, 4n=40). or aneupl0ids (n=6, 7, 17, 25, 53).     

Study on the Hybrid Endosperm Culture of Wheat-Rye In Vitro

The endosperm culture of wheat-rye hybrid was studied in order to explore a new pathway of chromosome engineering. The preliminary results were obtained to show that the endosperm callus formation could be induced from the young endosperm within 7–14 days after crossing on the medium supplemented with 2 ppm 2,4-D, 0.5 ppm kinetin and 3%–8% sucrose. The induction frequency of callus amounts to 35.3%. When the calli were transfered onto an auxin step-down medium containing 0.5 ppm IAA and 1 ppm kinetin, both shoots and roots were formed. 4 endosperm plantlets were obtained. The chromosome number in somatic cells of endosperm plantlets was very unstable. The numbers varied from 6—42, but there is no 49 to be found. The chromosome number with 1—4 times of 7 can be found in higher percentage.     

Studies on endosperm culture of Annona squamosa Linn

Mature endosperm tissue excised from germinated seeds (2–4 days after radicle emergence) of Annona squamosa grew and proliferated on White's basal medium supplemented with two cytokinins, an auxin and gibberellic acid. The callus obtained could be periodically subcultured. Shoot differentiation and root induction were obtained from callus on media of different compositions. Analyses of the root and young leaf tips showed triploid number of chromosomes (3n=21).Abbreviations Kin Kinetin - BAP 6-Benzylaminopurine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA naphthalene abetic acid - GA₃ Gibberellic acid Communication No. 3885     

In vitro growth of embryos and callus of coconut palm

Summary A medium for optimal growth of embryos of Jamaican Tall and Green Malayan Dwarf varieties of coconut palm was developed. The liquid basal Murashige and Skoog medium was supplemented with coconut milk, IAA and 2IP. Activated charcoal improved embryo growth on agar medium. A single callus line was initiated from solid endosperm and subcultured on basal Schenk and Hildebrandt medium supplemented with 2 mg per 1 NAA. Attempts at inducing organogenesis in the callus were unsuccessful. No vascular tissue was present. The callus was aneuploid with the chromosome number=8 (normal 2n=32). Florida Agricultural Experiment Stations Journal Series No. 542. The research was supported in part by the Horticultural Research Institute (to J. H. T.) and the American Philosophical Society (to J.B.F.).     

阳桃胚乳愈伤组织诱导和不定芽发生的研究

首次成功建立阳桃胚乳组织培养并获得胚乳再生植株。胚乳愈伤组织诱导以培养基MS 2,4-D2．0mgL^-1 BA0．2mgL^-1的效果最好,诱导频率可达94．7％,愈伤组织乳白色,结构致密,生长旺盛;将其接种在培养基MS ZT3．0mgL^-1 NAA0．2mgL^-1上,愈伤组织由乳白色致密型转变为淡绿色致密型,进而形成绿色芽点,分化出不定芽,分化频率可达73．3％;胚乳植株在培养基MS ZT2．0-2．5mgL^-1 NAA0．05mgL^-1上进行壮苗和营养繁殖。     

Callus Induction And Triploid Plant Regeneration From Endosprm of ‘Hongjiang’ Sweet Orange

Endosperm at late stage of cell formation, excised from open-pollinated fruit of sweet orange (Citrus sinensis Osbeck. cv.‘Hongjiang’), was suitable for culture in vitro. The results indicated that 2,4-D was necessary for callus' induction, and supplemented with BA, CH in medium was more effective: the percentage of induction was as high as 33.3%. Endosperm tissues, excised from the fruits treated with low temperature (4–7℃) for 16 and 19 days, and from the young seeds precuhured on MT basal medium respectively with NAA, GA3, BA for 2–6 days, also stimulated to callus formation. When endosperm callus was transfered to the differentiation medium, embryoids and shoot-buds only developed in a sequence of culture conditions. Callus was first cultured on MT+BA/GA3, then transfered to different media with various nitrogen or hormone concentration, and finally transferred back to the first culture medium. Shoots were regenerated from shoot-buds in the medium in presence of hormones with only BA or with GA3. The whole plants were regenerated from embryoids in presence of GA3 or with BA. Analysis of endosperm plantlets showed that 79.4% of the observed celld have chromosome number of 2n=26–27, nearly the triploid number of 2n=3x=27. Through grafting on lemon seedlings as rootstocks in vitro, plantlets were growing successfully in soil.     

玉米未成熟胚乳愈伤组织和器官的发生及其倍性变化的初步研究

本试验在附加和不附加外源激素的MS培养基上,均得到了玉米未成熟胚乳愈伤组织。愈伤组织在附加外源激素的MS培养基上达到器官分化,获得了发育正常的和许多畸形的胚乳植株。所得到的愈伤组织细胞和植株根尖细胞染色体数目和倍性是不稳定的,二者有相同的趋向,其中有整倍体的细胞(2n=10,20,30,40,50),也有各种非整倍体的细胞(2n=5—49)。     

Studies on Morphological Differentiation of Endosperm Plantlets of Chinese Gooseberry in Vitro

The present report deals with the process of embryoid induction and plantlet formation from cell-type endosperm of Actinidia chinensis var. chinensis and A. chinensis var. hispida. The callus was induced from endosperm on MS basic medium supplemented with zeatin 3 ppm, 2,4-D 0.5 ppm and CH 400 ppm and then transferred to differentiation medium of MS supplemented with zeatin 1 ppm and CH 400 ppm. After about half a month, the embryoid appeared from callus and then developed into plantlets. It could be seen from the histological figure 14—15, the embryoid of Chinese gooseberry is linear-shaped and consists of cells arranged in a long line in callus. When the cells regenerated at botk ends of the linear-shaped embryoid, the polarity of the embryoid is easily distinguished. The plantlets produced from embryoid appear rather stout at first and after some time, they changes gradually into normal plantlets.     

Triploid plant regeneration from immature endosperms of <Emphasis Type="Italic">Melia azedazach</Emphasis>

Melia azedazach, a plant for forestation, is popular in many countries. Development of triploid M. azedazach varieties will provide additional advantages, such as faster growth, higher biomass, and; therefore, increased productivity. In this study, we aimed to develop triploid M. azedarach L. by immature endosperm tissue culture. After 22 days of initiation of cultures, calli of the endosperm were visible. After 50 days cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l NAA and 1.0 mg/l BAP, maximum of callus induction rate from the immature endosperm with seed coat was obtained at 55.9%. The highest frequency of shoot induction from endosperm-derived callus was 98% and average of 16.7 shoots per explant on the medium supplemented with 1.5 mg/l BAP and 0.5 mg/l NAA after 42 days. A single shoot was detached from the multi-shoots and transferred to the rooting medium supplemented with 0.5 mg IBA, inducing root formation with 96.6% and with average of 5.8 roots per plantlet after 28 days. The plantlets transferred to polythene hycotrays containing soil and sand (mixture 1:1) in greenhouse showed 100% survival after transplantation. The endosperm-derived plantlets were 100% triploids as evidenced by flow cytometry analysis. Creating triploid M. azedazach plants by regenerating directly from endosperm (3n) described in this work required only 5 months whereas the traditional method of generating triploids through crossing between tetraploid (4n) and diploid (2n) plants could take up to 12 years.     

Induction and maintenance of friable callus from the cellular endosperm of Cocos nucifera L.

A fast-growing friable callus was initiated and maintained by subculture from the portion of coconut endosperm which was initially in contact with the zygotic embryo. Euwens' (Physiol. Plant. 36 (1976) 23) Y3 mineral formulation with kinetin (2 mg/l), high 2,4-dichlorophenoxyacetic acid (2,4,-D) (50 mg/l), and 1 g/l activated charcoal were used to initiate the callus in the dark. The callus could be subcultured on the basal medium with lowered 2,4-d levels and in the absence of charcoal. The success in obtaining the callus may be partly attributed to the use of explants excised in a sterile condition with the zygotic embryo in situ (no chemical sterilization involved), and the high auxin concentration in the medium. Preliminary cytological studies indicate a high degree of aneuploidy in the callus cells. Also, within the slow-growing callus cells oil globules had accumulated.     

Morphogenesis and plant regeneration from cultured endosperm of Emblica officinalis Gaertn

Mature endosperm of Emblica Officinalis (Euphorbiaceae) formed a continously growing callus on MS medium supplemented with an auxin (2,4-D or IAA) and a cytokinin (K or BAP). Subculture of callus on MS with BAP (0.2 mg/l) and IAA (0.1 mg/l) resulted in formation of shoots and embryo-like structures in 50 and 8 per cent cultures, respectively. Regeneration of shoots was more frequent when both BAP (0.2 mg/l) and IAA (0.1 mg/l) were present than on BAP (0.2 mg/l) alone. The embryo-like structures produced plantlets.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid - PDB para-dichlorobenzene (née Arora)     

Plant genotype and growth regulators interaction affecting in vitro morphogenesis of blackberry and raspberry

The morphogenic response of somatic (leaf and petiole) and de-differentiated tissue (callus) of two blackberry (Rubus fruticosus) and one raspberry (Rubus idaeus) cultivars have been studied in vitro. With the aim to induce regeneration the effect of two sets of plant growth regulator (PGR) combinations (high cytokinin/auxin ratios and high auxin/cytokinin ratios) in Murashige and Skoog basal medium, were analysed. The three cultivars were characterised by a qualitatively different morphogenic response to the PGR combinations. Raspberry adventitious shoot regeneration from somatic tissue was improved by the 6-benzylaminopurine (BAP)/indol-3-butyric acid (IBA) combinations. On the contrary, shoot regeneration of both blackberry cultivars was reduced by high concentrations of BAP and completely inhibited by BAP/IBA combination. Media supplemented with high auxin/cytokinin ratios promoted callus production and root differentiation according to genotype and type of auxin. All the genotypes responded to media supplemented with IBA. 2,4-dichlorophenoxyacetic acid induced good callus formation in blackberry, but was toxic to raspberry. Indirect shoot formation was observed only in callus of blackberry cultivar Hull Thornless cultivated on medium with 10 μM BAP, the same concentration able to trigger efficient direct shoot regeneration from leaf explants of the same cultivar. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of triploid plants from endosperm cultures of Phlox drummondii

Triploid plants of ornamental Phlox drummondii Hook. were raised from cultures of endosperm excised from immature fruits having zygotic embryo at early dicotyledonous stage. Endosperm tissue was firstly cultured with the embryo on the Murashige and Skoog’s (MS) medium supplemented with 5 μM 6-benzylaminopurine (BAP) + 10 μM α-napthaleneacetic acid (NAA) for 7 d and recultured after the embryo was removed. A friable callus appeared two weeks after removal of the embryo and it became compact callus mass in another three weeks. Upon transfer of this 5-week-old callus to the MS medium with 10 μM BAP + 2.5 μM indole-3-acetic acid (IAA), maximum percentage of green nodular shoot buds appeared from which regenerated dwarf shoots. Elongation of the dwarf shoots, however, required transfer of the individual dwarf shoots excised from the callus on the fresh medium and best results achieved on medium with low concentration of IAA (0.5 μM) in presence of 10 μM BAP. The shoots were then rooted in vitro and plants subsequently established in pots containing soil. Over 70 % of plants were triploid with a chromosome number of 2n=3x=21. Size of stem, leaves, flowers, pollen, and stomata of these triploid plants were higher and the plants were more vigorous as compared to naturally occurring diploid plants. In particular, flowers showed bright colour with enlarged central eye adding to their ornamental value.     

Morphogenic studies in tissue cultures of the parasiteSantalum album L.

Whole seeds, excised embryos, and excised endosperm ofSantalum album were aseptically cultured with a view to studying seed germination in isolation from the host species, and to establishing callus cultures from both embryo and endosperm for comparative studies et their morphogenesis. Seed germination and seedling formation occurred normally only on modified White's medium supplemented with casein hydrolysate or coconut milk, or with both substances. Neither the excised embryo nor the endosperm grew on any of the culture media tested. However in about 17 per cent seed cultures on White's medium supplemented with 2,4-D, kinetin, and yeast extract, the endosperm degenerated, whereas the embryo callused and subsequently differentiated into innumerable embryoids; eventually the embryoids developed into normal plantlets. Callusing of the endosperm occurred also in seed cultures on four media supplemented variously with 2,4-D, kinetin, and yeast extract. Although the endosperm tissue grew through several passages no organ fornation was observed.     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Production of triploid plants of papaya by endosperm culture

Triploid papaya (Carica papaya) plants were obtained by immature endosperm culture. Visible callusing of the endosperm occurred 21 days after initiation of cultures. A continuously growing callus was observed and a maximum of 68.7% of callus induction frequency was obtained when immature endosperm with embryo was cultured on Murashige and Skoog (MS) medium containing 6.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.5 μM α-naphthaleneacetic acid (NAA) and 4.0 μM 6-furfurylamino purine (Kn). Shoot buds were produced when the callus was subcultured on a medium supplemented with 6-benzyladenine (BA) or thidiazuron (TDZ) along with NAA. Shoots were detached from the callus and transferred to the elongation medium supplemented with indole-3-acetic acid (IAA) and BA. The combination of 3.0 μM IAA and 1.5 μM BA was the best in terms of the number of cultures (93.8%) showing axillary shoot proliferation. The addition of 2.0 μM indole-3-butyric acid (IBA) to the 1/2 MS medium was most effective at inducing root formation with 90% of the shoots developing four to five roots. Healthy rooted plantlets were transferred to pots containing sterilized bed soil and perlite (3:1) mixture in the greenhouse and 78% of the micropropagated plants survived transplantation. The leaves from endosperm-derived plants showed larger stomata and more chloroplasts in guard cells than that from the parent plants. Over 75% of the endosperm-derived plants were triploid with chromosome number 2n = 3x = 27.     

Embryological Studies on In Vitro Pollination in Rice

n vitro pollination and its embryological studies were carried out in two japonica cultivars of rice (Oryza sativa L.), “Chunjiang 05" and “95046". N6 basic medium supplemented with different exogenous hormones was used for ovary culture after in vitro pollination. The main results were as follows: (1) both cultivars were induced to set kernels after in vitro pollination. The frequency of seedset was 52.1%, including 28.4%normal embryo development, 2.2% abnormal embryo development and 21.5% callus formation. (2) The processes of embryo and endosperm development after in vitro pollination were basically as normal as those in vivo , except there was some retardation in the first division of zygotes and primary endosperm nuclei as well as in their subsequent development. However, both kernels and plantlets could be produced finally. (3) A few abnormal embryos were observed, for instance, proembryos with elongated suspensor and vacuolated proembryos. (4) Two types of calli in the cultured ovaries appeared, namely, the compact callus and the fragile callus, which were able to differentiate into adventitious buds and roots.     

Micropropagation of Sesbania aculeata (Pers.) by adventitious organogenesis

Multiple shoots differentiated from hypocotyl explants of Sesbania aculeata (Pers.) syn S. cannabina (Retz.) Pers., a leguminous woody shrub, when cultured on Murashige and Skoog's basal medium supplemented with auxin (IBA, NAA) or auxin and cytokinin (IBA + BAP, NAA + BAP). Shoot budding occurred directly from the explant as well as from callus. Differentiation of shoot and root occurred in one step in the same concentration of auxin or auxin and cytokinin. Elongation of shoots occurred in the shoot induction medium.     

Tissue culture and long-term regeneration of Phragmites australis (Cav.) Trin. ex Steud.

Phragmites australis tissue cultures were initiated from mature seeds on MS medium supplemented with 1 mgl^-1 each of 2,4-D and IAA. Cultures displayed typical embryogenic callus that was compact and bright yellow. Selection for embryogenic callus established long-term regenerable cultures. Removal of auxin from the basal medium allowed numerous complete plants to be recovered from the cultures. Histological study indicated both the presence of embryogenic-type cells and the bipolar development of regenerated plants.     

Plant regeneration from petal explants of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.