Micropropagation of guava (Psidium guajava L.)

Summary

Guava (Psidium guajava L.) is difficult to propagate using conventional asexual techniques, with most growers using seedling planting stock. However, these seedlings are highly variable. We therefore developed an in vitro technique to clonally propagate guava. Various concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate and micropropagate plants. Two explant sources were compared: greenhouse grown plants (GHRP) and in vitro-harvested axillary buds (IVDS). GHRP with BAP at 2 mg l^–1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm. Shoots 3.0 cm long were obtained with 0.5 mg l^–1 BAP, however only 2.1 shoots per explant were produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at 0.25 mg l–1 , with an average shoot length of 1.6 cm. Generally, lower concentrations of BAP gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and 2.0 cm, respectively) were obtained with 1 mg l^–1 indole-3-butyric acid (IBA). A protocol for producing clonal plants over eight weeks is described.     

Histology of somatic embryogenesis in mature tissues of olive (Olea europaea L.)

Summary

This anatomical investigation on olive secondary somatic embryos describes several aspects of embryo development, including proembryoid origin and growth, aspects of tissue differentiation, localization of somatic embryogensis, and starch occurrence. The failure of a number of secondary somatic embryos to develop into perfect structures is to be ascribed to defects in the last growth stages (fused embryos, fused cotyledons) and/or to tissue degeneration processes affecting both imperfect and apparently perfect somatic embryos.     

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.     

Silver nitrate and 2-isopentyladenine promote somatic embryogenesis in date palm (Phoenix dactylifera L.)

Embryogenic callus derived from offshoot tip of date palm (Phoenix dactylifera L.) was transferred to MS medium containing 0, 25, 50, 75, or 100 μM AgNO₃ combined with 0 or 0.5 μM 2iP. Embryogenic callus weight, number of embryos developed and embryo elongation were significantly influenced by the interaction between silver nitrate (AgNO₃) and 2iP. In the absence of 2iP, callus weight was greatest with 75 μM AgNO₃, but in the presence of 2iP omitting silver nitrate resulted in the highest callus proliferation. The number of embryos increased in response to increasing silver nitrate concentration in the absence of 2iP, but in the presence of 2iP increasing the concentration of silver nitrate gave the opposite trend. The number of resultant embryos was the highest on 25 μM AgNO₃in the presence of 0.5 μM 2iP. This treatment also caused maximum embryo elongation. The results have shown that silver nitrate promoted callus proliferation and enhanced the formation and elongation of somatic embryos of date palm. Furthermore, the action of silver nitrate was clearly modified by the addition of 2iP. Depending on the response, 2iP modification ranged from slight alteration to complete inversion of the general trend associated with increasing silver nitrate concentration. The observed stimulatory action of AgNO₃ on date palm somatic embryogenesis may contribute to improve existing regeneration systems particularly for recalcitrant date palm genotypes.     

Induction of somatic embryogenesis in lotus (Nelumbo nucifera Geartn.)

Callus induction and somatic embryogenesis of lotus (Nelumbo nucifera Gaertn.) cv. Satabankacha were studied. Callus was initiated by culturing bud, cotyledon, and young leaf explants on Murashige and Skoog (MS) (1962) medium containing a combination of 0, 4, 8 and 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 0, 1, 2 and 3 μM 6-furfuryl amino purine (kinetin) or substituting 0, 0.5 and 1 μM benzyladenine (BA) for kinetin. Bud explants cultured on medium containing 4 μM 2,4-D and 1 μM BA gave the best callus growth. For somatic embryogenesis, the calli initiated on MS medium containing a combination of 4, 6, 8 and 10 μM 2,4-D and 1 μM BA and subsequently transferred to media containing 2–4 μM 2,4-D and 0 or 0.5 μM BA produced the most somatic embryos. When cultures were 12-week-old, callus produced on medium with 6 μM 2,4-D and 1 μM BA showed the best growth for somatic embryo regeneration. When transferred to a medium with 2 μM 2,4-D and 0.5 μM BA somatic embryos were produced from 33% of the calli. Embryos developed to the stage proembryo within 4 weeks and formed globular, heart, torpedo and mature embryos within 16 weeks.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

Somatic embryogenesis and plant regeneration in black pepper (Piper nigrum L.): I. Direct somatic embryogenesis from tissues of germinating seeds and ontogeny of somatic embryos

Summary

A protocol was developed for induction, maturation and germination of somatic embryos from the tissues of germinating seeds of black pepper (Piper nigrum L.). Explants were cultured on growth regulator – free solid SH medium maintained in the dark. The first somatic embryos developing directly from the explant tissue were noticed after 60 d of culture. Somatic embryos originated from a ring-like tissue on the micropylar region of the seeds. Sucrose concentration of the medium was found to be crucial for the induction of somatic embryos, and 30 g l^–1 was found to be the optimum. Maturation and germination of somatic embryos were achieved on the same medium. Suspension culture enhanced the process of maturation and germination. Regenerated plants were established in soil. Histology confirmed the ontogeny and each stage of development. Growth regulators were found to inhibit the induction of somatic embryogenesis. Cytological analysis of the regenerated plants revealed the normal chromosome number of 2n=52.     

几种修剪树型对'珍珠'番石榴开花坐果及产量的影响

选用4年生'珍珠'番石榴为试脸材料,采用开心形、圆头形、平头形树型结构,以果树生产中采用的自然圆形树型结构为对照,研究4种树型修剪方法对'珍珠'番石榴新梢数、花蕾数、开花结果数及果实产量的影响.试验结果表明,'珍珠'番石榴树型圆头形、开心形促进开花坐果较好,圆头形有利于提高产量.     

Nutritional Status of Guava (Psidium Guqjava L.) Trees in North India

A survey of nutrient concentrations in guava leaves from trees with above average production was made in Uttar Pradesh. The values found were different from those published by other workers in respect of all macronutrients except potassium.     

改良CTAB法提取番石榴总DNA的初步研究

以番石榴为试材,采用CTAB、SDS和改良的CTAB法分别从新鲜的番石榴叶片中提取DNA,并对其进行琼脂糖凝胶电泳检测和SCoT-PCR分析.结果表明:3种方法均可以从番石榴的叶片中提取DNA,但改良的CTAB方法通过加入漂洗液2次洗涤,能够较好的去除样品中的多酚、多糖和蛋白质等物质,可以较好的从新鲜的叶片中提取较高质量的DNA,并可以用于SCoT-PCR标记分析.     

Direct somatic embryogenesis from explants obtained from in vitro germinated embryonic axes of Camellia sinensis (L.) O. Kuntze

Summary

Studies on direct somatic embryogenesis in several types of explant from in vitro plantlets of tea cultivar TRI 2025 were undertaken to select those most suitable to induce cotyledonary-type somatic embryos. Mature zygotic embryonic axes were surface-sterilised and cultured on MS medium without growth regulators containing 0.6% (w/v) agar. Results showed that 65% of embryonic axes that converted into plantlets at the fifth week of culture had succulent leaves. Several types of explant (normal leaves, large and small succulent leaves, hypocotyl segments and root tips) were isolated from in vitro plantlets at the fifth week and cultured on half-strength MS medium containing 2 mg l^–1 6-benzylaminopurine (BAP) and 0.2 mg l^–1 naphthalene acetic acid (NAA). Morphological and histological observations on somatic embryogenesis were made. The results indicated that somatic embryos were produced at high frequency (25 – 50%) directly from the surface of hypocotyl segments (HS) and large succulent leaves (LSL) after 6 weeks of culture. Efficient somatic embryogenesis was induced in small succulent leaves (SSL) after 16 weeks. Most somatic embryos originated directly from the cortical tissues of HS or the upper epidermal layers of SSL or LSL. HS and SSL from in vitro plantlets gave the highest production of typical, firm somatic embryos for use in tea improvement programmes and for in vitro conservation of tea germplasm.     

Somatic embryogenesis in cell suspension cultures of Prunus persica (L.)

Cell suspension cultures were derived from adventitious somatic embryos induced on cotyledons of rescued peach embryos. Embryogénie calli were induced and established on modified MS medium supplemented with 2,4-D, BAP, KN and inositol. Development of embryos during suspension cultures was identified by growth pattern and by histological and cytological examination, using acetocarmine stain. More embryogenic calli were produced using 2,4-D (5 (µM) than NAA. A gradual reduction of 2,4-D increased the recovery of globular embryos. Heart-stage embryos were developed on high-nitrogen salt medium, with added Ca(N03)2 (1000 mg I"1) and 6% sucrose. Somatic embryos turned green and sometimes red under white light. Embryo viability was highest when subculturing every 14 days. Viability of cells and differentiating embryos was assessed, microscopically, with Alexander’s stain. Viable embryos reacted pink to red while non- viable embryos reacted green to blue. Embryogenic cell suspension cultures were maintained for over ten months by regular subculturing after two weeks on modified MS medium with low 2,4-D, BAP and KN.     

Effect of selected amino acids and polyethylene glycol on maturation and germination of somatic embryos of guava (Psidium guajava L.)

Two selected amino acids (l-glutamine and l-proline) and polyethylene glycol (PEG) were evaluated for their effects on maturation and germination of somatic embryos in guava (Psidium guajava L.) cv. Banarasi local. Among the treatments of two amino acids (l-glutamine and l-proline) and PEG tested, 0.87 mM l-proline was most effective for maturation of somatic embryos. As compared to control, supplementation of PEG (1.0% or 2.0%) in maturation media showed significantly better maturation (increased maturation frequency). Lower stage somatic embryos showed prematuration germination with development of abnormal plantlets and only mature somatic embryos produced morphologically normal plantlets.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.     

Physiological Studies During the Growth and Development of Different Varieties of Guavas (Psidium Guajava L.)

In a study of three seed-bearing varieties of guava and one seedless variety changes in size and specific gravity and in the content of water, titratable acid, reducing and non-reducing sugars, tannins, pectin, and ascorbic acid were recorded at intervals from fruit-set to harvest. Respiration rates were also determined during this period.

Patterns of change were generally similar in seeded and unseeded varieties, though the sugars and ascorbic acid contents rose earlier and to higher levels in the seedless than in the seeded, and the fruits matured and fell from the trees earlier.

Increasing ascorbic acid content, concurrent with declining pectin content in the seeded varieties, is consistent with the hypothesis that pectin degradation and ascorbic acid are linked, but in the seedless variety a rapid rise in the ascorbic acid content occurred during a period of pectin accumulation. The end of the initial rapid decline in respiration rate generally coincided with the beginning of reducing-sugar accumulation.     

Improved efficiency of somatic embryogenesis from zygotic embryos in Hyoscyamus niger by seed water-soaking

We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somatic embryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the seeds in sterile distilled water for 16 h, prior to excision and culture of zygotic embryos, on MS basal medium supplemented with 2% sucrose, 2 g/l myo-inositol, 0.5 g/l 2-(N-morpholino) ethanesulfonic acid and 1 mg/l α-naphthalene acetic acid (NAA). The regenerated somatic seedlings were fertile and were morphologically uniform. This procedure is simple, rapid and effective for high frequency of plant regeneration via somatic embryogenesis. Moreover, this new method should facilitate the development of strategies to routinely transform recalcitrant plant species, including henbane.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L.

An efficient method of repetitive somatic embryogenesis and plant regeneration was established in Coriandrum sativum L. Embryogenic callus was induced from cotyledon and hypocotyl segments on Murashige and Skoog (MS) medium with 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), upon subculturing on medium having same level of 2,4-D at an interval of 3 weeks developed somatic embryos, which progressed to cotyledonary stage through early developmental stages of somatic embryogenesis. The transfer of somatic embryos at an early cotyledonary and cotyledonary stage in clumps in succession to fresh 4.52 μM 2,4-D supplemented medium developed embryos in a cyclic manner. Upon transferal to embryogenic clumps (cotyledonary embryos) to modified MS medium (4 g l⁻¹ KNO₃, 0.29 g l⁻¹ NH₄NO₃, 3 mg l⁻¹ thiamine HCl, 0.5 mg l⁻¹ pyridoxine HCl, and 5 mg l⁻¹ nicotinic acid), the embryos irrespective of the cycles underwent maturation and germination. Germinating embryos transferred to half-strength MS medium favored healthy growth of plantlets. The system of recurrent somatic embryogenesis in coriander offers a system for genes transfer and also scale-up production of modified plants.     

In vitro somatic embryogenesis from Mangifera indica L. callus

The nucellus and globular adventitious proembryos were removed from 2-month-old fruits of mango (Mangifera indica L.) cultivars ‘Ono’ and ‘Chino’, and were cultured on sterile, solid Murashige and Skoog (MS) medium that had been modified as follows: half-strength major salts and chelated iron; 20% (v/v) coconut water (CW); 6% sucrose; 100 mg l^?1 ascorbic acid and 400 mg l^?1 glutamine. Embryogenic explants were sub-cultured after 4–6 weeks in liquid modified MS medium containing 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) instead of CW. Rapidly growing cultures were established and were sub-cultured monthly. Somatic embryogenesis was induced following sub-culture from MS medium with 2,4-D to MS without growth regulators and with or without activated charcoal (0.5%). Germination of somatic embryos appeared to be enhanced by 1 mg l^?1 benzyladenine (BA); however, most of the germinating embryos became embryogenic.