CryoLetters 37 (4), 219-230 (2016) © CryoLetters, [email protected]

SEED DESICCATION TOLERANCE AND CRYOPRESERVATION OF PHILIPPINE CALAMANSI [x Citrofortunella microcarpa (BUNGE) WIJNANDS]

Olivia P. Damasco* and Luzminda C Refuerzo

National Plant Genetic Resources Laboratory-Institute of Plant Breeding-Crop Science Cluster, College of Agriculture, University of the Philippines Los Banos, College, 4031 Laguna, Philippines *Corresponding author email: [email protected]

Abstract BACKGROUND: The traditional on farm conservation of Calamansi [x Citrofortunella microcarpa (Bunge) Wijnands], an important indigenous Citrus species in the Philippines, is now being threatened by shifting agricultural crop production, climate change, and increasing biotic and abiotic stresses. OBJECTIVE: The study aimed to characterize the desiccation and cryopreservation tolerance of seeds as the basis for complementary long term ex situ conservation. MATERIALS AND METHODS: Intact seeds were desiccated in an airtight container filled with activated silica gel for 0-96 h. Seeds placed in cryotubes were subjected to rapid freezing in liquid nitrogen, rapid thawing in a water bath at 50 C for 3 min, and cultured on MS basal medium for seedling recovery and growth. Recovered seedlings were potted out in plastic bags filled with coir dust: garden soil mixture (1:1 v/v) and maintained in the nursery. RESULTS: Significant reduction in percentage germination was obtained at in a moisture content (MC) window between 24.3% and 4.2% and complete loss of viability at below 3.2%. The number of germinated embryos per seed was significantly reduced following desiccation from a mean of 4.2 embryos per seed for the untreated control to 1.2 to 1.02 embryos per seed at 33.3-4.2% MC, respectively. Recovery and germination of seeds after cryopreservation were obtained in a MC window between 24.3% and 4.2% with the maximum seed germination (27%) obtained at 13.4%. Germination abnormalities such as incomplete germination, greening and or enlargement of cotyledon without shoot emergence were observed in both desiccated and cryopreserved seeds. Variations in response to seed desiccation and cryopreservation were observed among Calamansi accessions tested. Maximum seedling recovery after liquid nitrogen storage varied between 12.5% and 61.5%. Recovered seedlings from desiccation and cryopreservation treatments survived ex vitro establishment and showed normal growth and similar morphology with the non-treated control seedlings. CONCLUSION: The partial tolerance of Calamansi seeds to desiccation and subsequent recovery after cryopreservation provides the basis for long term ex situ preservation of this valuable germplasm, although further optimization is needed. Keywords: seed desiccation, cryopreservation, Calamansi, Citrofortunella microcarpa

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INTRODUCTION conservation system is very costly and the number of accessions that can be preserved The Calamansi [x Citrofortunella is limited (25). Field conservation ex situ of microcarpa (Bunge) Wijnands], a shrub of Calamansi germplasm is limited and few the Rutaceae family, is an important accessions of living trees are available in the indigenous Citrus species in the Philippines. field collections in the Philippines, China, It is native in Asia, originally from China, Columbia, Malaysia, Spain, Thailand and and its cultivation has spread throughout the United States (33). Moreover, in the Philippines and Indonesia in Southeast Asia Philippines, is it mostly conserved in situ, on where the earliest descriptions were made farm: in farmers’ orchards, home gardens, (33). It is considered a natural intergeneric villages or in areas of major cultivation. hybrid between the loose skin mandarin This traditional way of conserving the crop orange (Citrus reticulata Blanco var. austere is now being threatened by climate change, Swingle) and Fortunella japonica (30, 31). pests and diseases, shifting agricultural Other English common names include practices and susceptibility to natural calamondin, calamandarin, golden lime, calamities. In addition, due to its short Philippine lime, Panama orange, Chinese productive periods (approximately 5-7 orange and acid orange (31, 33). years), plantations are now being replaced Calamansi is primarily valued for its by more profitable crops. These factors acid juice. It is commercially processed into slowly contribute to the loss of genetic bottled concentrate and ready-to-drink juice. diversity in Calamansi, hence the urgent The juice is also very popular as a flavor need for a complementary long term enhancer for native dishes in Philippines and conservation method. other Asian countries. Traditionally, it is The presence of high degree of used for medicinal and hygienic purposes polyembryony in most Citrus species (18, 33). It has a considerable amount of provides an opportunity to conserve the essential oils stored in the rind and is widely original genotype as seed despite the high used as a substitute to lemon or lime level of heterozygosity (21). However, essential oil. In other countries, it is very conservation of Citrus germplasm in seed popular as ornamental plant in gardens, in banks is difficult to apply because many pots and container gardens on terraces and species are classified as having intermediate patios, and as rootstock for lemon and oval seeds (17, 19), i.e. they can withstand partial kumquat for pot culture (23). dehydration but cannot be stored under Due to its varied uses, it is considered conventional genebank conditions because as one of the most important Citrus species they are cold sensitive and desiccation does in the Philippines with total production area not increase their longevity (8, 9, 16). For of about 20,245 ha and volume of intermediate and recalcitrant seeds and production of about 164,059 MT valued at asexually propagated species, 58.071M US$ (5). The Region IV B, cryopreservation or storage under liquid particularly the Oriental Mindoro province, nitrogen (LN) offers long term storage is the biggest producer. It is also grown capability, minimal storage space and throughout the country as a backyard crop. maintenance requirement, and maximal In most scientific institutions, Citrus stability of phenotypic and genetic behavior germplasm is traditionally conserved in of stored germplasm (11, 12, 13). At this clonal orchards and botanical gardens, while ultra-low temperature (-196ºC), all cellular genotypes of particular value are kept in divisions and metabolic processes are greenhouses and screen houses where they stopped, allowing conservation for unlimited can be protected from losses due to pest and periods of time (13, 26, 29, 35). For seeds diseases (19). However, this traditional that can withstand partial desiccation, seed

220 cryopreservation seems to be a promising Mature fruits of Calamandarin (Citrus long term ex situ technique, although high sp. variety Calamandarin), another native levels of recovery are species dependent and sour Citrus variety commonly grown in the low recovery is still observed in more country, were collected from a backyard desiccation-sensitive species (17). farm in Sta Maria, Laguna. In some reports, Cryopreservation of several poly and Calamandarin is also referred as another mono embryonic Citrus species has been English common name for Calamansi (31). applied to intact seeds, exised embryos and However, according to Citrus expert in the embryonic axes with varying rates of Philippines, Calamandarin is a unique success after dehydration to a MC range of variety different from Calamansi based on 17-5% depending on species (3, 4, 17, 19, some morphological features, and believed 20, 27, 36, 37). to be a natural hybrid between Calamansi To date, there are no established and mandarin, hence the name methods for long term preservation of Calamandarin (Coronel, personal Philippine Calamansi using the cryogenic communication). The taxonomy and exact technique. This study aimed to characterize origin of this variety is still unclear. the dessication and cryopreservation Calamandarin is also commonly used as tolerance of intact seeds of Calamansi as the rootstock for Calamansi and mandarin basis for long term conservation. seedlings. Specifically, it aimed to determine the The fruits were washed in soap and optimal moisture content window for water and the extracted seeds were washed successful cryopreservation and assess several times in running water to remove all variation in cryotolerance among accessions immature or partially developed seeds and tested. other adhering debris. Seeds were then surface-disinfected with 30% (v/v) solution MATERIALS AND METHODS of commercial bleach (sodium hypochlorite, 5% a.i) for 20 min, and rinsed thrice with Plant materials sterilized distilled water inside a laminar Physiologically mature fruits of flow hood cabinet. Calamansi were obtained from different localities in the provinces of Laguna, Seed desiccation and cryopreservation Batangas, and Oriental Mindoro in Southern Seeds were blotted briefly in sterile Luzon, and North Cotabato in Southern tissue paper and batches of 50 seeds were Mindanao. For the experiment on seed placed in 50 mm Petri dish without lids ande desiccation tolerance and cryopreservation, desiccated in an airtight glass jar container fruits were obtained from a local market in filled with activated silica gel (800 mL Bay, Laguna (14 11 N 11 17 E). While for capacity, 200 g silica gel, 100 seeds the response of other Calamansi accessions, approximately 13.7 g). Seed desiccation fruits were obtained from a backyard farm in was done inside the laboratory at 27-30 C Sta Maria, Laguna (14 28 12 N for 0 (control), 24-96 h at 12 h or 24 h 121 25 34 ), commercial farms in Rosario, interval depending on the experiment. The Batangas (13 51 N 121 12 E) and Socorro, seed MC at the end of each desiccation Oriental Mindoro (13 04 N 121 24 E), and a period was determined gravimetrically on 15 local market in Kabakan, North Cotobato seeds per treatment (3 replicates of 5 seeds) (7 07 N 124 49 E). The different following oven drying at 110°C for three accessions collected from different localities days and expressed on percentage fresh showed great variations in fruit and seed weight basis. After each desiccation period, sizes. The Oriental Mindoro and Batangas seeds were transferred immediately onto accessions had bigger fruits than the other recovery medium (control treatment) or to accessions. 2.0 mL (5 seeds per tube for Calamansi) or

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5.0 mL (10 seeds per tube for Calamandarin) randomly selected two year old seedlings cryotubes, plunged rapidly in LN, and kept maintained in the nursery. in storage for 48 h. Thereafter, seeds were thawed rapidly in a water-bath at 50°C for 3 Experimental design min, kept in the laminar flow hood for 1 h A total of 1,200 seeds extracted from and then transferred onto recovery medium. 400 fruits (Calamansi) and 750 seeds from 150 fruits (Calamandarin) were used for the Recovery and survival assessment experiments on seed desiccation tolerance The survival and recovery of seeds and the cryopreservation response. The from desiccation and cryopreservation experiments were laid out in Completely treatments were determined by culturing Randomized Design with three replications seeds on Murashige and Skoogs (MS) basal of 20 seeds per replicate per treatment. For medium supplemented with 30 g L-1 sucrose the response of the different accessions, 450 and solidified with 0.7% agar. Cultured seeds (150 fruits) per accession for Laguna, seeds were kept in a growth room at Batangas and Oriental Mindoro and 200 27±2°C, 16 h photoperiod and light seeds (100 fruits) for the North Cotobato irradiance of 65 µmol m-2 s-1 provided by accession were used. All percentage data two 40 watt fluorescent tubes. To hasten were arcsine-transformed prior to statistical germination, seed coats were removed after analysis using the Analysis of Variance. one week of culture. Final observations Comparison of treatment means was done on seed survival (all surviving seeds with or using the Least Significant Difference (LSD) without germination), germination at P=0.05. For the experiment on accession (emergence of both root and shoot), response, measurement values were germination abnormalities (incomplete expressed as means standard error of the germination either root or shoot emergence mean (SEM) obtained from 40-60 seeds per only, enlargement and/or greening of treatment depending on the accession. cotyledon without germination), number of Actual percentage values were presented in germinated seedlings per seed, shoot length, tables. root length and number of leaves of germinated seedlings were measured after RESULTS six weeks of culture. Seedlings recovered from desiccation Response to desiccation and cryopreservation treatments obtained No significant differences in percentage from seeds collected from Laguna province survival were observed between fully were transplanted in pots (1 L capacity) hydrated seeds (control) and those containing coconut coir dust: garden soil desiccated to as low as 13.4% MC (Table 1). mixture (1:1, v/v). For the first eight weeks Further desiccation to 6.9% and 4.2%, after transplanting, seedlings were however, showed significantly reduced maintained in the greenhouse under natural percentage survival at 70% and 40%, light conditions and ambient temperature respectively. Complete loss of seed viability (30 C ± 2 C). Established seedlings were was observed at below 3.0% MC. In terms transferred to bigger pots (3 L capacity) of percentage germination (emergence of filled with garden soil and kept in an open both shoot and root), no significant nursery under ambient temperature and differences were observed between the natural day length for further growth and control and seeds desiccated at 33.3% MC development. Plants were maintained (Table 1). However, further desiccation to regularly following the recommended below 24.3% resulted in much significantly procedures. Measurements for reduced percentage germination: 71.8% at morphological parameters were made on 24.3% MC and 12.1% at 4.2% MC, respectively. The critical seed MC was observed at 13.4% and percentage 222 germination was not significantly different The post-thaw survival and complete with 24.3% MC. The decrease in seed seedling recovery of cryopreserved seeds germination at reduced MC was were observed only at a MC range of 24.3% accompanied by an increase in germination to 4.2% (Table 1). The maximum post-thaw abnormalities such as shoot or root percentage survival of frozen seeds (27.9%) emergence only, and enlargement and or was observed at 6.9% MC although this was greening of the cotyledon but without not significantly different to 13.4% MC germination (Table 1). The percentage (25%). The lowest percentage survival germination abnormality was, however, the (2.8%) was observed at 4.2% MC which was same for all desiccation treatments and not significantly different to 24.3% MC ranged from 6.9-11.4% (Table 1). (10.6%). Although percentage survival was Nonetheless, at 6.9% and 4.2% MC, highest at 6.9% MC, germination was germination abnormalities were mostly significantly highest (25%) at 13.3%, limited to greening and or enlargement of followed only by 6.9 % MC (15.4%) and the cotyledon but without germination much significantly reduced germination at (Table 1). 24.3% and 4.2% MC. Seeds subjected to cryopreservation showed much lower Response to cryopreservation percentage germination compared with

Table 1. Percentage survival, germination and germination abnormality in desiccated (-LN) and cryopreserved (+LN) Calamansi seeds.

MC (% Desiccation treatment Cryopreservation treatment ± (-LN) (+LN) SEM) Survival Germination Germination Survival Germination Germination (%) (%) abnormality (%) (%) abnormality (%) (%) 54.72 100.0 a 100.0 a 0 0 c 0 c 0 ±2.07 40.6 100.0 a 88.6 a 11.4* 0 c 0 c 0 ±1.34 33.5 100.0 a 86.2 a 13.8* 0 c 0 c 0 ±0.99 24.3 82.7 a 71.8 b 10.9* 10.6 bc 7.1 bc 3.5* ±0.59 13.4 70.4 ab 59.8 b 10.6** 25.8 a 23.3 a 2.5* ±0.41 6.9 40.0 bc 30.1 c 6.9*** 27.9 a 15.4 b 12.5*** ±0.58 4.2 22.8 c 12.1 c 10.7*** 2.8 c 2.8 c 0 ±0.16 3.1 0c 0c 0 0c 0c 0 ±0.29

Desiccation time ranged from 0, 24-96 h at 12 h interval. Survival includes all surviving seeds with or without germination. Germination includes seedlings with both root and shoot emergence. Germination abnormality includes incomplete germination either root or shoot only*, greening of cotyledons** or enlargement and greening of cotyledons***. Means in a column with the same letter are not significantly different at P=0.05 using LSD. All values are means of three replicates of 20 seeds per treatment measured after 6 weeks in culture.

223 desiccation treatment only (Table 1). At A significant reduction in the mean 13.3% MC, germination was 59.8%, and number of germinated seedlings per seed following cryopreservation, this was reduced was observed after cryopreservation (Table to 23.3%, an almost 50% reduction in 2). However, the same negative effect was germination. Similarly, at 6.9% MC, about observed with desiccated seeds even before a 50% reduction in germination was cryopreservation, suggesting that the observed for cryopreserved seeds. reduction in number of germinated seedlings Calamansi seeds were sensitive to was mostly due to the desiccation treatment cryopreservation as shown by a (Table 2). At reduced MC, the shoot length corresponding decrease in germination of and number of leaves were more or less the desiccated seeds after cryopreservation. same for with or without cryopreservation The germination abnormality after treatment (Table 2). Mean shoot length cryopreservation was similar with the ranged from 46.66 - 85.45 mm while mean desiccation treatment only suggesting that number of leaves ranged from 1.0-1.5. these abnormalities were mostly due to However, root length was very much damages in dehydrated tissues. The optimal inhibited after cryopreservation (range of MC for cryopreservation was 13.3% while 32.5 - 83.0 mm) compared with the the highest and lowest MC limits were desiccation treatment only (range of 67.0 - 24.3% and 4.2%, respectively. 132.25 mm).

Table 2. Growth parameters measured from germinated seedlings of desiccated (-LN) and cryopreserved (+LN) Calamansi seeds.

MC (% Desiccation treatment Cryopreservation treatment ± SEM) (-LN) (+LN) No. of Shoot Root No. of No. of Shoot Root No. of GE/S length length leavesNS GE/S length length leavesNS (mm)NS (mm)NS (mm)NS (mm)NS 54.72 4.2 a 44.9 130.5 2.9 0 - - - ±2.07 40.6 2.7 b 48.4 138.8 2.9 0 - - - ±1.34 33.5 1.2 c 41.a 149.4 2.1 0 - - - ±0.99 24.3 1.2 c 56.4 132.3 3.6 1.5 66.3a 32.5 2.0 ±0.59 13.4 1.02 c 53.1 138.2 2.1 1.2 85.5 52.5 2.8 ±0.41 6.9 1.08 c 51.3 144.2 2.2 1.2 79.9 46.7 2.2 ±0.58 4.2 1.03 c 55.9 67.0 3.0 1.0 46.7 83.0 2.8 ±0.16 3.1 0 ------±0.29

Desiccation time ranged from 0, 24-96 h at 12 h interval, GE/S-germinated embryos per seed. Means in a column with the same letter are not significantly different at P=0.05 using LSD, NS-not significantly different at P=0.05. All values are means taken from uneven number of germinated seedlings measured after 6 weeks of culture.

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Table 3. Percentage germination of desiccated (-LN) and cryopreserved (+LN) Calamandarin seeds.

MC (% Germination (%) ± SEM) Desiccation (-LN) Cryopreservation (+LN) 50.60±2.18 100 a 0c 24.46±2.82 100 a 0c 12.59±2.7 85.1 ±10.66 a 27.9±4.14 a 5.14±0.58 85.6±9.41 a 18.2±5.69 ab 2.69±0.21 36.3±7.75 b 12.5±3.41 b

Desiccation times ranged from 0, 24-96 h at 24 h interval. Germination includes seedlings with both root and shoot emergence. Means in a column with the same letter are not significantly different at P=0.05 using LSD. All values are means ±SEM of three replicates of 20 seeds per treatment

(critical MC of 5.15%) than Calamansi seeds Response of Calamandarin and Calamansi (critical MC of 13.6%) and showed accessions to desiccation and complete loss of viability at 3.1% (Table 1 cryopreservation and Table 3). Seed germination from Calamandarin seeds could be desiccated cryopreserved seeds was obtained in a MC to as low as 5.14% MC without a significant window of 12.59% to 2.69%, with the reduction in germination, while significantly maximum post-thaw germination (27.9%) reduced germination (36.3%) was observed obtained at 12.69% MC. Similar to at 2.69% MC (Table 3). Calamandarin Calamansi (Table 1), the germination of seeds exhibited greater desiccation tolerance desiccated Calamandarin seeds after

Table 4. Initial seed characteristics and germination of desiccated (-LN) and cryopreserved (+LN) seeds from four Calamansi accessions.

Accession Fresh seed MC Germination (%) weight (% ± SEM) (g/20 seeds) Desiccation Cryopreservation ±SEM (-LN) (+LN) Laguna 2.62± 0.034 52.7 ±2.25 100 0 13.54±0.79 57.3± 22.7 26.6±9.7 5.38±0.56 6.7±3.67 0 Batangas 2.89 ±0.081 67.5 ±1.51 100 0 14.25±0.07 59.3±4.6 24.4±7.5 4.19±0.14 10±2.5 0 Oriental 3.00 ±0.050 57.5±1.24 100 0 Mindoro 15.94±0.02 64.9±7.91 25.4±4.8 4.41±0.12 18.3±11.7 0 *(greening of cotyledon) North Cotobato 2.10±0.016 59.38± 0.53 100 0 4.67±0.02 72.5±2.5 61.5±5.5

Desiccation time in silica gel ranged from 0, 62 and 72 h. Fresh weight of seeds was measured in intact seeds and expressed as means ± SEM for three replicates of 20 seeds. MC (%) is expressed as means ± SEM of three replicates of five seeds. Germination includes seedlings with both root and shoot emergence and expressed as means ±SEM of two (North Cotobato accession) or three replicates of 20 seeds per treatment. 225

Table 5. Ex vitro survival and morphological characteristics of two year old seedlings recovered from desiccated (-LN) and cryopreserved (+LN) Calamansi seeds.

Morphological parameter Untreated -LN +LN control Survival ex vitro (%) 100 80 60 Plant height (mm) 101 ± 10.38 88.6 ± 4.77 90 ± 11.03 Plant canopy (mm) 106.75 ± 10.21 105.5 ± 10.49 111.2 ± 16.53 Stem diameter (mm) 12.25 ± 1.11 13.4 ± 0.87 17.5 ± 1.32 No. of secondary branches 7.5 ± 2.1 9.4 ± 3.04 13.0 ± 2.88 Leaf length (mm) 26.33 ± 0.98 24.7 ± 1.41 25.6 ± 0.84 Leaf width (mm) 12.97 ± 0.41 11.5 ± 0.63 13.93 ± 0.58 Other juvenile Presence of Presence of Presence of characteristic thorns thorns thorns

Measurements were taken from seedlings recovered from desiccated (-LN) and cryopreserved (+LN) seeds at 13.5-6.9% MC from Laguna accession (Table 1 and Table 4). All values are means ±SEM taken from 30 (Untreated control and -LN) and 20 (+LN) seedlings. cryopreservation was significantly reduced response was limited to greening and from 85.1% to 27.9%, 85.6% to 18.21%, and enlargement of the cotyledon. In contrast, 35.3% to 12.5% at 12.59%, 5.14%, and the North Cotobato accession at 4.44%MC 2.69% MC, respectively (Table 3). Although had almost the same germination for the Calamandarin had greater seed desiccation desiccation control and following tolerance than Calamansi, both were equally cryopreservation treatment. The results sensitive to cryopreservation (Table 1 and showed that seeds collected from different Table 3) provinces exhibited varied responses to Calamansi accessions collected from desiccation and cryopreservation suggesting various provinces showed variations in fresh possible differences in the seeds collected. seed weight, initial seed MC, and tolerance to desiccation and cryopreservation (Table Ex vitro establishment of seedlings from 4). At 13.54-15.94% MC, germination was cryopreserved seeds almost similar for all accessions and ranged Complete seedlings were recovered from 57.3% (Laguna accession) to 62.9% from cryopreserved seeds in a MC window (Mindoro Oriental accession). At MC range of 24.3% to 4.2%. However, only those of 4.4- 5.38%, germination was greatly recovered from 13.5% to 6.9% MC survived reduced to 18.3% (Mindoro accession) and soil establishment. The seedlings recovered 6.7% (Laguna accession). The accession from cryopreserved seeds at higher (24.3%) from North Cotobato, however, showed high or lower (4.2%) MC limit, were generally germination (61.7%) even after drying to weak and did not survive soil transplanting. 4.41% MC. Similarly, at 13.54-15.94% The two year old seedlings recovered from MC, all Calamansi accessions showed more desiccation and cryopreservation treatments than 50% reduction in germination after showed normal growth and morphology cryopreservation; from 57.3% to 26.6% for similar to non-treated control seedlings the Laguna accession, 59.3% to 24.4% for (Table 5). Batangas accession, and 62.91% to 18.75% for Mindoro Oriental accession, DISCUSSION respectively. At a much reduced MC (4.19- 5.38%), recovery after cryopreservation was Calamansi seeds partially tolerated nil for Batangas and Laguna accessions desiccation and could be considered while for the Mindoro accession the intermediate in terms of seed storage

226 behavior category (8, 9). The percentage of the seed coat one week after culture seed germination was significantly reduced further enhanced seed germination. to almost 50% at 13.54% MC and much Following desiccation, the number of significantly reduced germination at 6.9- germinated embryos was reduced 4.2% MC. Total loss of seed viability significantly from 4-6 to 1-2 embryos per occurred below 3.0% MC. The decrease in seed. At reduced seed MC, the smaller germination was due to an increase in the embryos within the seed were severely dried occurrence of germination abnormalities up compared to the bigger ones and only such as shoot or root emergence only and those vigorous and big embryos which enlargement and/or greening of the tolerated desiccation germinated in vitro. cotyledon without germination. The results The decrease in the number of germinated of this study supported the earlier findings of embryos could be due the varied desiccation Ellis et al (10) on the intermediate seed rates of the embryos within the seed, i.e. storage behavior of this species grown in smaller embryos were dehydrated faster than Vietnam. However, the percentage the bigger embryos. This observation is germination obtained in the present study similar to earlier report on other was lower (59.8-12.1% at 13.54-4.2% MC) polyembryonic Citrus species wherein compared to their earlier report (75-15% at dehydration procedure resulted in a reduced 15-5% MC). germination of apomictic seedlings and Intermediate seeds can tolerate partial allowed germination of zygotic seedling desiccation to about 7% to 12% MC (19). Responses of intact seeds of Citrus depending on the species, but further drying species to desiccation varied depending on leads to more rapid loss in viability and species, genotype/varieties within the immediate damage occurs on further species, seed size as a factor of seed desiccation (8, 9). These seeds cannot be maturity, and even the drying method used stored under conventional genebank (19). conditions because they are cold sensitive Partial dehydration of tissues can offer and desiccation does not increase their protection against intra-cellular ice longevity (8, 9). Other Citrus species, for formation during rapid freezing in LN (17, example C. deliciosa, behaved similarly and 19). The state of water and the osmotic showed 50% germination after drying to equilibrium related to movements of water 15% MC (19). In contrast, seeds of other into and out of the cells are parameters of Citrus species could be desiccated to a particular importance for cryopreservation moisture level of 16% (C. sinsensis), 10% (22). Recovery of cryopreserved seeds of (C. limon) (19) and 8-6% (C. medica) (3) Calamansi was observed at a seed moisture with negligible reduction in germination window of 24.3-4.2% with maximum post- levels. In the present study, Calamandarin thaw germination (25%) obtained at 13.3% seeds likewise showed insignificant MC. The wide range of MC window for reduction in germination after drying to cryopreservation could be due to differences 12.59-5.14% MC. in the rate of dehydration and subsequent Calamansi, being highly polyembryonic cryotolerance of the embryos within the produced about 4-8 nucellar embryos of seed. The desiccated seeds subjected to varying sizes and stages of development cryopreservation showed an almost 50% within the seed. In vitro culturing on reduction in germination compared with the nutrient medium allowed almost all desiccation treatment only (from 59.3% to embryos, even the small ones, to germinate 23.3% and 30.1% to 15.4% at 13.4% and completely, but at different times. 6.4% MC, respectively). Furthermore, the nutrient medium supported Similarly, in C. grandis (17), the the growth of seedlings even those with germination of seeds exposed to LN at some germination abnormalities. Removal optimal MC was significantly lower than

227 that obtained from desiccation treatment seeds varied between 18.7% and 61.5% at only. The further drop in germination of 15.94-4.19% MC. The observed wide range desiccated seeds after LN exposure indicated in cryotolerance among the accessions tested that rapid freezing had some detrimental warrant further studies. The extent of genetic effects on seed germination (9). A decline diversity in Calamansi is currently unknown in seed tolerance to LN exposure at low and, as reported, there is only one water content as earlier reported was due to commercial variety grown in the country sensitivity of lipid rich seed (Citrus being (24). In other species, factors contributing to high in lipids) to rapid cooling; and that lipid wide variations in desiccation tolerance and rich seeds did not withstand the presence of cryotolerance include: inherent genetic freezable water in their tissues during the variation, seed geographic origin, and seed cooling/thawing process (17). In oily seeds, maturity. Great variability in seed the interaction between storage lipids and desiccation and cryopreservation tolerance water somehow promoted formation of ice existed among Citrus species such as in: C. crystals large enough to cause lethal damage limon (19) C. aurantium (19), C. medica (4, (34). On the other hand, other Citrus 21) C. madurensis (3), C. sinsesis (20), C species such as C. sinensis, C. madurensis madurensis (3) C. grandis (17, 36) C. and C. aurantifolia showed almost similar paradisa (37), C. aurantifolia (3, 19 21), germination levels for desiccated and Australian wild Citrus species C. cryopreserved seeds suggesting that LN australasica, C inodora, and C. garrawayi treatment did not cause any additional (15), C deliciosa (19) and detrimental effects on desiccated seeds (17). genotypes/varieties within C. grandis (36). In the present study, the number of The maximum seedling recovery reported germinated embryos per seed and for these species varied between 22-93%. In occurrence of germination abnormalities in the case of coconut zygotic embryos, cryopreserved seeds were similar with the experiments on different coconut varieties desiccated seeds only. The results suggest (tall, dwarf and hybrids) led to post-thaw that the decrease in number of germinated recoveries of 10% to 93% (1, 2, 27). nucellar embryos per seed and the However, in Coffea arabica (32) and Acer occurrence of germination abnormalities pseudoplatanus (7), seed geographic origin were mostly due to tissue damages from contributed to the differences in tolerance to desiccation and that no additional damages desiccation and cryopreservation. It was occurred from LN treatment. However, root previously reported that seeds from different elongation was very much inhibited in geographic gradients might have differences germinated seedlings from cryopreserved in proportion of saturated fatty acid, leading seeds. to differences in mean onset temperature for The study also showed that lipid melting (14). Goveia et al (14) Calamandarin seed, although more tolerant suggested that the developmental stage of to desiccation compared with Calamansi the seed is a critical factor when selecting (85% germination at 12.59-5.14% MC), was seed axes for cryopreservation. Similarly, quite sensitive to cryopreservation. At coconut zygotic embryos excised from seed 12.59% MC, the germination after of different maturities showed varied cryopreservation was reduced considerably cryotolerance with the best results obtained from 85.1% to 27.9%. from seeds before full maturity (1, 2). Calamansi accessions collected from Successful cryopreservation was also several provinces in Southern Luzon and reported using immature seeds of Mantisia Southern Mindanao showed varying spathulata and M. wengeri (6). The present responses to seed desiccation and study provided initial data on the cryopreservation. The maximum post-thaw cryotolerance among accessions tested. germination obtained from cryopreserved However, there is a need for further

228 investigation using a larger number of seed 4. Cho EG, Normah MN, Kim HH, samples and more accessions to confirm this Ramanatha Rao V & Engelmann F result. Emphasis should be placed on seed (2002) CryoLetters 23, 309-316. geographic origin, seed maturity, season of 5. DA-BAS Department of Crop Statistics harvest, and genetic characteristic of the for the Philippines (DA-BAS) (2013) seed source. http://www.bas.gov.ph. Accessed March The present study provided information 2014. on seed desiccation tolerance and 6. Das Bhowmik SS, Kumaria S & Tandon cryopreservation as an initial basis for the P (2011) CryoLetters 32, 498-505 long term preservation of Calamansi 7. Daws MI & Pritchard HW (2008) germplasm. The successful recovery of CryoLetters 29, 189–198, 527. seedlings from cryopreserved seeds 8. Ellis RH, Hong TD & Roberts EH followed by successful establishment in the (1988) Annals of Botany 61, 405-408. nursery is the first step towards maximizing the opportunities for the cryopreservation of 9. Ellis RH, Hong TD & Roberts EH this important Citrus species. Being partially (1990) J. Exp Bot 41, 1167–1174. tolerant to desiccation, seed 10. Ellis RH, Mai-Hong T, Hong TD, Tan cryopreservation is a promising long term ex TT, Xuan-Chuong ND, Hung LO, Ngoc- situ conservation strategy. Being the first Tam B &Le-tam VT (2007) Seed report on seed cryopreservation of Science & Technology 35, 460-476. Philippine Calamansi, further experiments 11. Engelmann F (1997) Plant Genetic are needed to improve the cryopreservation Resources Newsletter 112, 9-18. response. Investigations on the rate of 12. Engelmann F (2000) In cooling rate (controlled versus rapid cooling Cryopreservation of Tropical Plant used in the present study) and the causal Germplasm: Current Research Progress factors contributing to wide variation in seed and Application, (eds) Engelmann F, desiccation tolerance and cryopreservation Takagi H JIRCAS, Tsukuba/IPGRI, from accessions or geographic origins are Rome, pp 8-20. needed in protocol development. 13. Engelmann F (2004) In Vitro Cell Dev B 40, 427-433. Acknowledgements: Research funding 14. Goveia M, Kioko JI & Berjak P (2004) from the Institute of Plant Breeding-Crop Seed Science Research 14, 241-248. Science Cluster, University of the 15. Hamilton KN, Ashmore SE & Pritchard Philippines Los Banos, College, Laguna, HW (2009) CryoLetters 30, 268-279. Philippines and Research grant from 16. Hong TD, Linington S & Ellis RH ACIAR-Bioversity for the initial research on (1996) Seed Storage Behaviour: a Citrus seed cryopreservation is gratefully Compendium. Handbooks for Genebanks ackowledged. Technical support was No. 4, International Plant Genetic provided by Ms Flora Cuevas and Marcelina Resources Institute, Rome. Tejano. 17. Hor YL, Kim YL, Ugap A, Chabrillange N, Sinniah UR, Engelmann F & Dussert REFERENCES (2005) Annals of Botany 95, 1153-1161. 18. Ladion HG (1985) Healing Wonders of 1. Assy-Bah B & Engelmann F (1992) Herbs. Philippine Publishing House, CryoLetters 13, 67-74. Manila, Philippines. 126 pp. 2. Assy-Bah B &Engelmann F (1992) 19. Lambardi M, de Carlo A, Biricolti S, CryoLetters 13,117-126. Puglia AM, Lombardo G. Siragusa M & 3. Cho EG, Kim HH, Baek HJ, Gwag JG & de Pasquale F (2004) CryoLetters 25, Normah MN. (2003) J Kor Soc Hort Sci 81-90. 44, 565-568. 229

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