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Cryopreservation of shoot tips of recalcitrant and tropical species: Advances and strategies

Normah MN, Sulong N, Reed BM

Cryobiology, 2019 04;87:1-14.
PMID: 30677412 DOI: 10.1016/j.cryobiol.2019.01.008

There is a pressing need for practical and successful conservation efforts to establish long-term germplasm collections of recalcitrant and tropical species, given the challenge and threat that these plants are facing. Cryopreservation is the only way of conserving some of these species, especially those with temperature or desiccation sensitive (recalcitrant) seeds. This review covers reports on cryopreservation studies of shoot tips (apical and axillary) of tropical and subtropical plants. Since many of these species have recalcitrant seeds, the cryopreservation successes, failures and problems involved with these seeds are also discussed. The methodologies, important factors and steps involved in successful cryopreservation protocols are analyzed. Finally strategies are suggested to develop a successful cryopreservation protocol for new plant species, in particular those with tropical recalcitrant seeds.

Matched MeSH terms: Seeds/physiology
Fulltext Stimulatory effects of gamma irradiation on phytochemical properties, mitotic behaviour, and nutritional composition of sainfoin (Onobrychis viciifolia Scop.)

Mohajer S, Taha RM, Lay MM, Esmaeili AK, Khalili M

ScientificWorldJournal, 2014;2014:854093.
PMID: 25147870 DOI: 10.1155/2014/854093

Sainfoin (Onobrychis viciifolia Scop. Syn. Onobrychis sativa L.) is a bloat-safe forage crop with high levels of tannins, which is renowned for its medicinal qualities in grazing animals. Mutagenesis technique was applied to investigate the influence of gamma irradiation at 30, 60, 90, and 120 Gy on mitotic behavior, in vitro growth factors, phytochemical and nutritional constituents of sainfoin. Although a percentage of plant necrosis and non-growing seed were enhanced by irradiation increment, the germination speed was significantly decreased. It was observed that gamma irradiated seeds had higher value of crude protein and dry matter digestibility compared to control seeds. Toxicity of copper was reduced in sainfoin irradiated seeds at different doses of gamma rays. Anthocyanin content also decreased in inverse proportion to irradiation intensity. Accumulation of phenolic and flavonoid compounds was enhanced by gamma irradiation exposure in leaf cells. HPLC profiles differed in peak areas of the two important alkaloids, Berberine and Sanguinarine, in 120 Gy irradiated seeds compared to control seeds. There were positive correlations between irradiation dose and some abnormality divisions such as laggard chromosome, micronucleus, binucleated cells, chromosome bridge, and cytomixis. In reality, radiocytological evaluation was proven to be essential in deducing the effectiveness of gamma irradiation to induce somaclonal variation in sainfoin.

Matched MeSH terms: Seeds/physiology
Desiccation sensitivity and cryopreservation of excised embryonic axes of Citrus suhuiensis cv. limau madu, Citrumelo [Citrus paradisi macf. × Poncirus trifoliata (l.) raf.] and Fortunella polyandra

Al Zoubi OM, Normah MN

Cryo Letters, 2012 May-Jun;33(3):241-51.
PMID: 22825791

Excised embryonic axes from seeds of three taxa, namely, Citrus suhuiensis cv. limau madu, Citrumelo (Citrus paradisi x Poncirus trifoliate) and Fortunella polyandra, were desiccated in a laminar airflow, over silica gel, and ultra-rapidly. Desiccation sensitivity (WC50) was estimated for each taxon using the quantal response model. High desiccation tolerance (WC50 = 0.11 g water per g dry mass. g/gdw) was observed for limau madu embryonic axes desiccated in a laminar airflow and ultra-rapidly (WC50 =0.10 g/gdw). Desiccation tolerance was substantially lower (WC50 = 0.19 g/gdw) for silica gel dehydration. Similarly, high desiccation tolerance (WC50 = 0.15 g/gdw) was associated with F. polyandra embryonic axes when desiccated in a laminar airflow, while a lower desiccation tolerance (WC50 = 0.17 g/gdw) was observed with silica gel dehydration. Ultra-rapid desiccation led to the highest desiccation tolerance (WC50 = 0.14 g/gdw). The dehydration rate, however, had no influence on desiccation tolerance (WC50 ~ 0.14 g/gdw) for Citrumelo embryonic axes. After each desiccation period, embryonic axes were directly immersed in liquid nitrogen (LN) followed by rapid rewarming. Normal seedling recovery of 80 to 83% for excised embryonic axes of limau madu was observed for laminar airflow and ultra-rapid dehydration, but for silica gel dehydration, 57% recovery was obtained. Similarly, for Citrumelo, high recoveries of 100% and 97% were obtained from axes desiccated in a laminar airflow and using ultra-rapid dehydration, respectively, whereas a lower value was associated with silica gel dehydration (80%). For F. polyandra, 50% recovery was obtained both for laminar airflow and ultra-rapid dehydration, while much lower recovery (43%) was associated with silica gel dehydration. Regardless of the drying method employed, axis survival percentages following exposure to LN were commensurate with the desiccation sensitivity pattern.

Matched MeSH terms: Seeds/physiology
Optimal hydration status for cryopreservation of intermediate oily seeds: Citrus as a case study

Hor YL, Kim YJ, Ugap A, Chabrillange N, Sinniah UR, Engelmann F, et al.

Ann Bot, 2005 Jun;95(7):1153-61.
PMID: 15781438

The purpose of this study was to investigate the basis of the optimal hydration status for cryopreservation of intermediate oily seeds using Citrus as a model.

Matched MeSH terms: Seeds/physiology*
Cryopreservation of whole seeds and excised embryonic axes of Citrus suhuiensis cv. limau langkat in accordance to their desiccation sensitivity

Makeen MA, Noor NM, Dussert S, Clyde MM

Cryo Letters, 2005 Jul-Aug;26(4):259-68.
PMID: 19827255

Following the investigation of desiccation sensitivity and freezing tolerance of the whole seed of Citrus suhuiensis cv. limau langkat, desiccation sensitivity and cryopreservation of the excised embryonic axes from the seeds of the same species were examined. Three drying conditions were employed: desiccation by equilibrium for the whole seeds and desiccation in laminar airflow and over silica gel for the excised embryonic axes. The relevance of desiccation sensitivity (WC50) to cryopreservation of whole seeds and excised axes was investigated. High desiccation tolerance (WC50 = 0.034 g H2O x g(-1)dw) was acquired for axes desiccated with faster dehydration rate (1.5 g x g(-1) x h(-1)) in laminar airflow compared to substantially lower desiccation tolerance (WC50 = 0.132 and 0.110 g H2O x g(-1)dw) acquired under slower dehydration rates (1.0 and 0.005 g x g(-1) x h(-1)) for axes desiccated over silica gel and whole seeds desiccated by equilibrium respectively. While few whole seeds (8.3%) survived freezing, high recovery percentages of axes (83.3% and 62.2%) after freezing were obtained under laminar airflow and silica gel drying conditions respectively. Irrespective of the drying method employed, axes survival percentages after exposure to LN temperature commensurate with the desiccation sensitivity pattern. For the whole seeds, a factor other than desiccation sensitivity that limits the tolerance to exposure to LN temperature seems to exist and still needs to be defined.

Matched MeSH terms: Seeds/physiology*
Fulltext Mixed Mating System Are Regulated by Fecundity in Shorea curtisii (Dipterocarpaceae) as Revealed by Comparison under Different Pollen Limited Conditions

Tani N, Tsumura Y, Fukasawa K, Kado T, Taguchi Y, Lee SL, et al.

PLoS One, 2015;10(5):e0123445.
PMID: 25938512 DOI: 10.1371/journal.pone.0123445

The maintenance of mixed mating was studied in Shorea curtisii, a dominant and widely distributed dipterocarp species in Southeast Asia. Paternity and hierarchical Bayesian analyses were used to estimate the parameters of pollen dispersal kernel, male fecundity and self-pollen affinity. We hypothesized that partial self incompatibility and/or inbreeding depression reduce the number of selfed seeds if the mother trees receive sufficient pollen, whereas reproductive assurance increases the numbers of selfed seeds under low amounts of pollen. Comparison of estimated parameters of self-pollen affinity between high density undisturbed and low density selectively logged forests indicated that self-pollen was selectively excluded from mating in the former, probably due to partial self incompatibility or inbreeding depression until seed maturation. By estimating the self-pollen affinity of each mother tree in both forests, mother trees with higher amount of self-pollen indicated significance of self-pollen affinity with negative estimated value. The exclusion of self-fertilization and/or inbreeding depression during seed maturation occurred in the mother trees with large female fecundity, whereas reproductive assurance increased self-fertilization in the mother trees with lower female fecundity.

Matched MeSH terms: Seeds/physiology
Resource use of insect seed predators during general flowering and seeding events in a Bornean dipterocarp rain forest

Nakagawa M, Itioka T, Momose K, Yumoto T, Komai F, Morimoto K, et al.

Bull. Entomol. Res., 2003 Oct;93(5):455-66.
PMID: 14658448

Insect seed predators of 24 dipterocarp species (including the genera ot Dipterocarpus, Dryobalanops and Shorea) and five species belonging to the Moraceae, Myrtaceae, Celastraceae and Sapotaceae were investigated. In a tropical lowland dipterocarp forest in Sarawak, Malaysia, these trees produces seeds irregularly by intensely during general flowering and seeding events in 1996 and/or 1998. Dipterocarp seeds were preyed on by 51 insect species (11 families), which were roughly classified into three taxonomic groups: smaller moths (Trotricidae, Pyralidae, Crambidae, Immidae, Sesiidae, and Cosmopterigidae), scolytids (Scolydae) and weevils (Curdulionidae, Apionidae, Anthribidae, and Attelabidae). Although the host-specificity of invertebrate seed predators has been assumed to be high in tropical forests, it was found that the diet ranges of some insect predators were relatively wide and overlapped one another. Most seed predators that were collected in both study years changes their diets between general flowering and seeding events. The results of cluster analyses based on the number of adult of each predator species that emerged from 100 seeds of each tree species, suggested that the dominant species was not consistent, alternating between the two years.

Matched MeSH terms: Seeds/physiology
Seed dispersal and spatial pattern in tropical trees

Seidler TG, Plotkin JB

PLoS Biol, 2006 Oct;4(11):e344.
PMID: 17048988

Theories of tropical tree diversity emphasize dispersal limitation as a potential mechanism for separating species in space and reducing competitive exclusion. We compared the dispersal morphologies, fruit sizes, and spatial distributions of 561 tree species within a fully mapped, 50-hectare plot of primary tropical forest in peninsular Malaysia. We demonstrate here that the extent and scale of conspecific spatial aggregation is correlated with the mode of seed dispersal. This relationship holds for saplings as well as for mature trees. Phylogenetically independent contrasts confirm that the relationship between dispersal and spatial pattern is significant even after controlling for common ancestry among species. We found the same qualitative results for a 50-hectare tropical forest plot in Panama. Our results provide broad empirical evidence for the importance of dispersal mode in establishing the long-term community structure of tropical forests.

Matched MeSH terms: Seeds/physiology*
Fulltext Impacts of logging on density-dependent predation of dipterocarp seeds in a South East Asian rainforest

Bagchi R, Philipson CD, Slade EM, Hector A, Phillips S, Villanueva JF, et al.

Philos Trans R Soc Lond B Biol Sci, 2011 Nov 27;366(1582):3246-55.
PMID: 22006965 DOI: 10.1098/rstb.2011.0034

Much of the forest remaining in South East Asia has been selectively logged. The processes promoting species coexistence may be the key to the recovery and maintenance of diversity in these forests. One such process is the Janzen-Connell mechanism, where specialized natural enemies such as seed predators maintain diversity by inhibiting regeneration near conspecifics. In Neotropical forests, anthropogenic disturbance can disrupt the Janzen-Connell mechanism, but similar data are unavailable for South East Asia. We investigated the effects of conspecific density (two spatial scales) and distance from fruiting trees on seed and seedling survival of the canopy tree Parashorea malaanonan in unlogged and logged forests in Sabah, Malaysia. The production of mature seeds was higher in unlogged forest, perhaps because high adult densities facilitate pollination or satiate pre-dispersal predators. In both forest types, post-dispersal survival was reduced by small-scale (1 m(2)) conspecific density, but not by proximity to the nearest fruiting tree. Large-scale conspecific density (seeds per fruiting tree) reduced predation, probably by satiating predators. Higher seed production in unlogged forest, in combination with slightly higher survival, meant that recruitment was almost entirely limited to unlogged forest. Thus, while logging might not affect the Janzen-Connell mechanism at this site, it may influence the recruitment of particular species.

Matched MeSH terms: Seeds/physiology*
Studies on seed germination and growth in weed species of rice field under salinity stress

Hakim MA, Juraimi AS, Hanafi MM, Selamat A, Ismail MR, Karim SM

J Environ Biol, 2011 Sep;32(5):529-36.
PMID: 22319865

An investigation was made to see the salt tolerance of 10 weed species of rice. Properly dried and treated seeds of weed species were placed on 9 cm diameter petridishes lined with Whatman No. 1 filter paper under 6 salinity regimes, viz. 0 (control), 4, 8, 16, 24 and 32 dS m(-1). The petri dishes were then kept in germinator at 25 +/- 1.0 degrees C and 12 hr light. The number of germinated seeds were recorded daily. The final germination percentage, germination index (GI), seedling vigour index, mean germination time and time for 50% germination were estimated. Root and shoot lengths of the weed seedlings were measured at 20 days after salt application and relative growth values were calculated. Results revealed that salinity decreased final germination percentage, seed of germination as measured by GI, and shoot and root length in all the species. Germination of most of the weed seeds was completely arrested (0) at 32 dS m(-1) salinity except in E. colona (12%) and C. iria (13.9%). The species C. iria, E. colona, J. linifolia and E. crusgalli showed better germination (above 30%) upto 24 dS m(-1) salinity level and were regarded as salt-tolerant weed species. J. linifolia, F. miliacea, L. chinensis and O. sativa L. (weedy rice) were graded as moderately tolerant and S. zeylanica, S. grosus and C. difformis were regarded as least tolerant weed species.

Matched MeSH terms: Seeds/physiology
Complex pollination of a tropical Asian rainforest canopy tree by flower-feeding thrips and thrips-feeding predators

Kondo T, Nishimura S, Tani N, Ng KK, Lee SL, Muhammad N, et al.

Am J Bot, 2016 Nov;103(11):1912-1920.
PMID: 27797714

PREMISE OF THE STUDY: In tropical rainforests of Southeast Asia, a highly fecund thrips (Thrips spp.) responds rapidly to the mass flowering at multiple-year intervals characteristic of certain species such as the canopy tree studied here, Shorea acuminata, by feeding on flower resources. However, past DNA analyses of pollen adherent to thrips bodies revealed that the thrips promoted a very high level of self-pollination. Here, we identified the pollinator that contributes to cross-pollination and discuss ways that the pollination system has adapted to mass flowering.
METHODS: By comparing the patterns of floral visitation and levels of genetic diversity in adherent pollen loads among floral visitors, we evaluated the contribution of each flower visitor to pollination.
KEY RESULTS: The big-eyed bug, Geocoris sp., a major thrips predator, was an inadvertent pollinator, and importantly contributed to cross-pollination. The total outcross pollen adhering to thrips was approximately 30% that on the big-eyed bugs. Similarly, 63% of alleles examined in S. acuminata seeds and seedlings occurred in pollen adhering to big-eyed bugs; about 30% was shared with pollen from thrips.
CONCLUSIONS: During mass flowering, big-eyed bugs likely travel among flowering S. acuminata trees, attracted by the abundant thrips. Floral visitation patterns of big-eyed bugs vs. other insects suggest that these bugs can maintain their population size between flowering by preying upon another thrips (Haplothrips sp.) that inhabits stipules of S. acuminata throughout the year and quickly respond to mass flowering. Thus, thrips and big-eyed bugs are essential components in the pollination of S. acuminata.

Matched MeSH terms: Seeds/physiology
The quality of barley husk-caryopsis adhesion is not correlated with caryopsis cuticle permeability

Brennan M, Paterson L, Baharudin AAA, Stanisz-Migal M, Hoebe PN

J Plant Physiol, 2019 Dec;243:153054.
PMID: 31648109 DOI: 10.1016/j.jplph.2019.153054

Adhesion of the barley husk to the underlying caryopsis requires the development of a cuticular cementing layer on the caryopsis surface. Differences in adhesion quality among genotypes have previously been correlated with cementing layer composition, which is thought to influence caryopsis cuticle permeability, the hypothesised mechanism of adhesion mediation. It is not yet known whether differences in adhesion quality among genotypes are determined by changes in caryopsis cuticle permeability. We examined changes in candidate cementing layer biosynthetic and regulatory genes to investigate the genetic mechanisms behind husk adhesion quality. We used both commercially relevant UK malting cultivars and older European lines to ensure phenotypic diversity in adhesion quality. An ethylene responsive transcription factor (NUD) is required for the development of the cementing layer. To examine correlations between gene expression, cementing layer permeability and husk adhesion quality we also treated cultivars with ethephon (2-chloroethylphosphonic acid) which breaks down to ethylene, and silver thiosulphate which inhibits ethylene reception, and measured caryopsis cuticle permeability. Differential adhesion qualities among genotypes are not determined by NUD expression during development of the cementing material alone, but could result from differences in biosynthetic gene expression during cementing layer development in response to longer-term NUD expression patterns. Altered caryopsis cuticle permeability does result in altered adhesion quality, but the correlation is not consistently positive or negative. Cuticle permeability is therefore not the mechanism that determines husk adhesion quality, but is likely a consequence of the required cuticular compositional changes that determine adhesion.

Matched MeSH terms: Seeds/physiology*
Fulltext Determination of suitable microspore stage and callus induction from anthers of kenaf (Hibiscus cannabinus L.)

Ibrahim AM, Kayat FB, Hussin ZE, Susanto D, Ariffulah M

ScientificWorldJournal, 2014;2014:284342.
PMID: 24757416 DOI: 10.1155/2014/284342

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.

Matched MeSH terms: Seeds/physiology*
High frequency plant regeneration from mature seed of elite, recalcitrant Malaysian indica rice ( Oryza sativa L.) CV. MR 219

Sivakumar P, Law YS, Ho CL, Harikrishna JA

Acta. Biol. Hung., 2010 Sep;61(3):313-21.
PMID: 20724277 DOI: 10.1556/ABiol.61.2010.3.7

An efficient in vitro plant regeneration system was established for elite, recalcitrant Malaysian indica rice, Oryza sativa L. CV. MR 219 using mature seeds as explant on Murashige and Skoog and Chu N6 media containing 2,4-dichlorophenoxy acetic acid and kinetin either alone or in different combinations. L-proline, casein hydrolysate and L-glutamine were added to callus induction media for enhancement of embryogenic callus induction. The highest frequency of friable callus induction (84%) was observed in N6 medium containing 2.5 mg l(-1) 2,4-dichlorophenoxy acetic acid, 0.2 mg l(-1) kinetin, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate, 20 mg l(-1) L-glutamine and 30 g l(-1) sucrose under culture in continuous lighting conditions. The maximum regeneration frequency (71%) was observed, when 30-day-old N6 friable calli were cultured on MS medium supplemented with 3 mg l(-1) 6-benzyl aminopurine, 1 mg l(-1) naphthalene acetic acid, 2.5 mg l(-1) L-proline, 300 mg l(-1) casein hydrolysate and 3% maltose. Developed shoots were rooted in half strength MS medium supplemented with 2% sucrose and were successfully transplanted to soil with 95% survival. This protocol may be used for other recalcitrant indica rice genotypes and to transfer desirable genes in to Malaysian indica rice cultivar MR219 for crop improvement.

Matched MeSH terms: Seeds/physiology

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