Artificial Plant Seeds and Their Application

Yilong HOU Zhiruo QI Sihan YU Chuiyu KONG Tongning BAI Qing LI

Abstract  The artificial plant seed is a new technology developed on the basis of the  in vitro  plant culture technology. As a high-tech achievement with great development potential and economic value in the 21  st  century， artificial plant seeds have great potential advantages. This paper briefly introduced artificial plant seeds， their composition and advantages， the preparation of artificial plant seeds and the application of artificial plant seeds.

Key words  Artificial seeds; Advantages; Applications

Received： October 8， 2021  Accepted： November 11， 2021

Supported by Dalian Science and Technology Innovation Project （2019J13SN120）.

Yilong HOU （1963-）， male， professor， PhD， devoted to research about plant biotechnology.

*Corresponding author.

Concept， Composition and Advantages of Artificial Plant Seeds

Concept of artificial plant seeds

Artificial seeds or synthetic seeds are artificially-made seeds. The artificial seed technology is an emerging technology developed on the basis of  in vitro  plant culture technology， which has been included in the "Eureka" plan in Europe. The research on artificial seeds in China is at the forefront of the world， and the research on artificial seeds is listed as "863" high-tech development plan. Artificial seeds are artificial capsules containing plant embryoid bodies or buds， nutrients， hormones and other ingredients. The concept of artificial seeds was first proposed by Murashige at the 4  th  International Plant Tissue Cell Culture Conference in 1978. Murashige believes that with the continuous development of plant tissue culture technology， a small number of explants can be used to simultaneously cultivate a large number of embryos. These embryos are embedded in a certain kind of capsules to make them have the function of seeds and can be directly sown in the field. In 1985， Japanese scholar Kamada extended the concept of artificial seeds. He believes that granules formed by embeding meristems （buds， calli， embryoid bodies and growth points，  etc. ） produced by tissue culture through appropriate methods that can develop into complete plants and can replace natural seeds for sowing， can be called artificial seeds. In 1995， Chinese scientists Chen  et al.  further expanded the concept of artificial seeds. They believe that granules formed with somatic embryos produced in  in-vitro  plant culture or meristems （such as embryoids， buds and stem segments） that can develop into complete plants and are embedded in shells that contain nutrients and have protective functions and capable of germinating under suitable conditions， can be called artificial seeds. In recent years， the concept has developed from the narrow sense of coating somatic embryos to the coating of any suitable plant propagules， such as adventitious buds， tubers， axillary buds， bud tips， protocorms， calli and hairy roots.

Composition of artificial plant seeds

A complete artificial seed consists of three parts： artificial seed coat， artificial endosperm， and artificial seed embryo  （embryoid）.

① Artificial seed coat： It is a colloidal compound film wrapped around the outermost layer of artificial seeds. Such film can not only allow the smooth exchange of internal and external gas， but also prevent the leakage of water and various nutrients in artificial endosperms. In addition， it should have a certain degree of mechanical resistance to pressure.

② Artificial endosperm： It refers artificially prepared nutrients needed to ensure the growth and development of artificial  embryos.

③ Artificial seed embryo： Artificial seed embryos are the propagules （living part） of artificial plant seeds， which are equivalent to embryos of natural seeds. Therefore， they are the core component of artificial seeds. They can be divided into two categories： somatic embryos and non-somatic embryos. Somatic embryos are structures with embryos， radicles， and natural seed embryos produced by tissue culture. Non-somatic embryos include adventitious buds， axillary buds， stem buds， protocorms， hairy roots， calli，  etc.  Artificial seed embryos have the ability to germinate and grow into plants.

Advantages of artificial seeds

As a high-tech achievement with great development potential and economic value in the 21  st  century， artificial seeds have following advantages：

① The use of artificial seeds for rapid propagation is convenient for transportation and storage， and is not restricted by environmental factors. Factory production and propagation can be carried out all year round.

② Artificial endosperms can be formulated according to the requirements of different plants （for example： proper amount of nutrients， hormones， pesticides， antibiotics and herbicides can be added）， so as to facilitate the healthy growth of embryoid bodies.

③ Embryoid bodies are produced by artificial asexual reproduction， which is conducive to the preservation of excellent traits of plants.

④ The use of artificial seeds can fix heterosis and speed up the breeding of plant varieties.

⑤ The use of artificial seeds can preserve precious plant  varieties.

⑥ The use of artificial seeds can reproduce some plants that do not bear fruit under natural conditions or whose seeds are very expensive.

⑦ Embryoid bodies produced by plant tissue culture have the characteristics of large number， fast reproduction speed and complete structure.

⑧ The use of artificial seeds can artificially control crop growth and resistance.

⑨ The use of artificial seeds can overcome harmful microorganisms such as viruses accumulated by some plants due to long-term vegetative reproduction.

⑩ Artificial seeds allow direct seeding and mechanized  operation.

Preparation of Artificial Plant Seeds

Preparation of embryoid bodies and their simultaneous growth

① Low temperature method： Low temperature hinders the synthesis of tubulin and the formation of the spindle apparatus is blocked， so the number of cells remained in the metaphase of mitosis increases.

② Inhibitor method： DNA synthesis inhibitors were added， such as 5-aminouracil，  etc.  at the beginning of cell culture， so that cell growth basically stops in G1 phase.

③ Ventilation method： Studies have found that injecting nitrogen or ethylene 1 to 2 times a day in the cell suspension culture medium for a few seconds or longer each time can significantly increase the mitotic synchronization rate.

Since it is difficult to obtain embryoid bodies of many plants， and the use of embryoid bodies to produce artificial seeds is affected by high frequency variation， the commercial application prospects of artificial seeds are greatly restricted. With the development of tissue culture technology and embedding technology， embedding bodies are no longer limited to embryoid bodies. For example， buds sucking， protocorms， shoot tips， internodes and axillary buds can be used as embedding propagules for artificial seed production. The rich sources of embedded propagules not only reduce the production cost of artificial seeds， but also speed up the research and development of artificial seeds.

Preparation of artificial endosperm

It is roughly similar to the culture medium of cells and tissues. Commonly used artificial endosperm is MS （or SH， White） medium plus potato starch hydrolysate. It is also possible to add appropriate amount of hormones， antibiotics， pesticides， herbicides，  etc.  to the above-mentioned medium as needed to facilitate the growth and survival of embedded propagules， but these additives should be non-toxic to embedded propagules and will not produce any impact on the growth of plants in variation.

Preparation of embedding agents

The embedding technology is one of the most critical steps in the production process of artificial seeds， and it has extremely important effects on the germination rate， plantlet formation rate and survival rate after storage. Therefore， the embedding technology has always been a research hotspot and one of the fastest growing fields. The materials used for embedding include sodium alginate， potassium alginate， carrageenan， sodium alginate added with gelatin， sodium pectin， carboxymethyl cellulose，  etc.  Among them， sodium alginate is the most widely used.

Embedding

First， 4% sodium alginate is usually prepared and then added with embryoid bodies in a certain proportion. After mixing， it is dripped into the 2.0%-2.5% CaCl2 solution. After 10 to 15 min of ion exchange complexation， round artificial seeds with certain rigidity are formed. Then， they are rinsed with sterile water for 20 min to terminate the reaction. The seeds are picked up and air-dried.

Application of Artificial Seeds

Since the concept of "artificial seeds" was proposed， there have been many research reports on this field at home and abroad， and especially， the application of artificial seed technology in the production of cash crops has produced greater economic effects. In recent years， the scope of research on artificial seeds has become wider and wider， not only on food crops and economic plants， but also on ornamental plants， submerged plants， and important and rare medicinal plants.

Pinellia ternata  Briet

P. ternata  is a perennial herb of the Araceae family. It is used as a medicine with tubers and has the effect of moistening lung for arresting cough. It is used frequently. Traditionally， wild tubers are mainly excavated， but  P. ternata  is a weedy plant， and due to the development of barren hills and slopes， the impact of disastrous climate， the widespread use of chemical fertilizers， pesticides and herbicides and artificial over-excavation， wild  P. ternata  resources are depleted day by day. In addition， because of its low reproduction coefficient and asexual reproduction methods using tubers and bulbils during cultivation，  P. ternata  has been seriously infected with viruses， and the yield and quality in field cultivation are low. People are not motivated to plant  P. ternata  and the supply of  P. ternata  is becoming less and less. However， domestic and foreign demand for  P. ternata  has been increasing. Especially， with global warming and increasing lung diseases， its price has also risen year by year. He  et al.   [1]  and Zhang  [2]  have done some useful researches on artificial seeds of  P. ternata ， and found that artificial seeds made from small tube tubers could form commercial products in the same year， the yield was twice that of cultivated seedlings， and effective alkali content increased by 1.6 times. Zhang  et al.   [3]  established a  P. ternata  tuber suspension system by converting solid culture to liquid suspension culture， and used small tubers as propagules of artificial seeds to study the effects of artificial seed coat matrixes， artificial endosperm  components，  storage conditions and germination matrixes on the germination rate and seedling rate of artificial seeds. The results showed that the artificial seeds made with 4.0% sodium alginate+2.0% CaCl2+2.0% chitosan as an artificial seed coat matrix and 1/2 MS liquid medium+0.2 mg/L NAA+0.1 mg/L GA3+0.5 mg/L BA+0.4 mg/L penicillin+0.3% carbendazim powder+0.2% sodium benzoate+1.0% sucrose+0.5% activated carbon as artificial endosperm components had the highest germination rate and seedling rate. The germination rate and seedling rate of such artificial seeds after 20 d storage at 4 ℃ were 82.8% and 78.6%， respectively， and with the increase of storage temperature and storage time， the germination rate and seedling rate of artificial seeds dropped significantly. Mao  [4]  used small tubers of  P. ternata  obtained by suspension culture differentiation as an embedding material to study the effects of different concentrations of chitosan， polyethylene glycol， carbendazim and sodium benzoate on the germination rate and seedling rate of artificial seeds of  P. ternata . The results showed that when the artificial seed coat of  P. ternata  was 3% sodium alginate+2% CaCl2+0.05% CoCl2+0.05% CuCl2+0.1 mg/L GA3+4% chitosan+2% polyethylene glycol+0.4% sodium benzoate， the germination rate was the largest at 85%; and when the artificial seed coat of  P. ternata  was 3% sodium alginate+2% CaCl2+0.05% CoCl2+0.05% CuCl2 +0.1 mg/L GA3+4% chitosan+4% polyethylene glycol +0.2%   carbendazim， the seedling rate was the highest at 95%.

Dendrobium candicum  Wall. ex Lindl.

Dendrobium candicum  Wall. ex Lindl. is a perennial epiphytic herb of the genus  Dendrobium  of the Orchidaceae family. For thousands of years， it has been listed as top-grade Chinese medicine with  Panax ginseng， Ganoderma lucidum， Cordyceps sinensis ，  etc. ， and it is hailed as "the first of the nine immortal herbs in China" in the medical classic "Dao Zang" of the Tang Dynasty. Due to its slow growth cycle and endangered wild resources， it was listed as a rare and endangered medicinal plant under national key protection in 1987. The emergence of the artificial seed technology provides a new way for the industrialization of  D. candicum . In 2011， Zhang  et al.   [5]  established a method for making artificial seeds of  D. candicum  using protocorms as propagules， and investigated the effects of endosperm and seed coat components on germination and seedling formation of the artificial seeds. In 2012， Zeng  et al.   [6]  designed 36 kinds of artificial endosperm using sucrose， starch， ABA， NAA， activated carbon， carbendazim， and sodium alginate and studied their effects on germination and growth of artificial seeds of  D. candicum  using ungerminated protocorms and germinated protocorms of  D. candidum  as propagules. They screened out appropriate propagule and endosperm formula， and then made coating on this basis.  The results showed that the artificial seeds with germination protocorms as propagules had a higher germination rate and survival rate. The suitable artificial endosperm combination was sucrose 3.0%， ABA 1.0 mg/L， activated carbon concentration 0.5%， carbendazim  3.0% ， and sodium alginate 3.0%. Coating the outer layer of the artificial  D. candidum  seeds could obtain higher germination rate and survival rate under natural conditions. In 2013， Lai  [7]  added different concentrations of activated carbon， sodium thiosulfate and polyvinylpyrrolidone to a liquid culture medium to study their effects on the proliferation， browning and differentiation of   D. candidum   protocorms， then made artificial seeds using protocorms obtained from proliferation by embedding with sodium alginate and calcium nitrate and studied the effects of different artificial seed coat formulas， basic media and carbon sources， carbendazim and activated carbon on germination of the artificial seeds， as well as the effect of abscisic acid on storage of the artificial seeds. In 2016， Zhou  [8]  design artificial seed coat matrixes using  D. candidum  tissues after 30 d of callus differentiation and germination and sodium alginate， CaCl2， penicillin， streptomycin and carbendazim powder as materials， and studied the effects of artificial endosperm components on germination rate of the artificial seeds. The results showed that the artificial seeds made from 4% sodium alginate+0.2 mol/L CaCl2+0.4 mg/L penicillin+0.4 mg/L streptomycin+0.3% carbendazim powder as the artificial seed coat matrix and 1/2MS+0.5 mg/L NAA+1.5 mg/L KT+1.5 mg/L 6-BA as artificial endosperm had the highest growth rate and germination rate.

Yilong HOU  et al.  Artificial Plant Seeds and Their Application

Taxus cuspidata  S. et Z.

T. cuspidata ， also known as yew， is a first-level national key protected plant. In recent years， people have isolated an anti-cancer active ingredient—paclitaxel from roots， stems and leaves of  T. cuspidata . Studies have shown that paclitaxel can bind to tubulin and promote its polymerization， and inhibit the mitosis of cancer cells and prevent the proliferation of cancer cells， and it has obvious effects of resisting leukemia， lung cancer， ovarian cancer， and breast cancer. According to analysis， the branches and leaves of  T. cuspidata  contain the highest paclitaxel among the 4 yew varieties in China.  Taxus  plants have a low seed germination rate under natural conditions. In addition， the reproduction speed of  Taxus  is slow because of being dioecious and low natural seed setting rate， few germplasm resources， long seed dormancy and other reasons， so propagating  Taxus  from natural seeds is very limited. Compared with the propagation method through natural seeds， artificial seeds have the advantages of fast propagation and saving money， manpower and land. Therefore， it is of great significance to the protection and medicinal value of  Taxus  species.  Sun  et al.   [8]  used the calli and axillary buds of a variety of  T. cuspidate  ‘Napa as propagules， and carried out initial research on the production process， endosperm formulation and germination conditions of its artificial seeds. They prepared the artificial seeds by adding different types and concentrations of soluble starch， elicitors and plant hormones to a sodium alginate gel system by the instillation method. The results showed that the reaction of 3.0% sodium alginate and 2.5% CaCl2 for 8-10 min gave the best seed coat for artificial seeds; the addition of 1.5% soluble starch could improve the water retention of artificial endosperm， and the water loss rate after storage at 24 ℃ for 15 d was 25.29%; adding elicitors to the artificial endosperm achieved the best effect when 10  -6  mg/L salicylic acid was used， and the seed particles were uniform and transparent; and adding NAA to artificial endosperm could increase the germination rate of artificial seeds， and 1.5 mg/L NAA achieved the best effect， with the germination rate reaching 38% after 30 d of culture. It can be seen from the research that the sodium alginate embedding systems have poor water retention， so that water-soluble nutrients can be quickly leached， and the artificial seeds made can only be stored for a short period of time， and are often blocked when they germinate. It is believed that future research should find a solid embedding matrix that is more suitable for the practical application of artificial seeds.

Cotton

Cotton is one of the main economic crops in China， and conventional varieties currently produced are prone to biological confounding due to their pollination characteristics. Although hybrids have yield advantages， they require during the seed production process manual emasculation and pollination， which is expensive and greatly limits the production and utilization of upland cotton seeds. Cotton is one of the crops more difficult to reproduce asexually. Artificial seeds have broad application prospects in the rapid propagation of clones， fixation of heterosis and combination with genetic engineering. Therefore， if a feasible method of asexual reproduction of cotton can be found， it will open up an economical and effective way for the preservation of the pure state of cotton excellent germplasms and the utilization of hybrids. In 1995， Zhang  et al.   [9]  used 3-day-old sterile seedling hypocotyls of upland cotton Kezi 201 as explants， and induced somatic embryogenesis on the MS solid medium added with 0.1 mg/L 2，4-D， 0.2 mg/L IAA and 0.1 mg/L KT. They selected mature cotyledon embryos for the production of artificial seeds， and found that 2.0% sodium alginate was a reasonable embedding medium concentration developed for cotton artificial seeds， and the artificial seeds formed by embedding had uniform particle size and high germination rate. Under aseptic conditions， the seedling rate of the artificial seeds reached 40.0%. In 2016， Hu  et al.   [9]  used a new upland cotton variety "Zhongmiansuo 47" as explants， embedded the explants with sodium alginate+MSB， and coagulated them with a CaCl2 solution to make artificial endosperm beads， and they then embedded the artificial endosperm beads using a higher concentration of sodium alginate added with different concentrations of methylcellulose and solidified them with a CaCl2 solution to obtain artificial seed coat， so as to study the germination effect of adding the antibacterial agent in the artificial seed coat of the upland cotton vegetative buds on their germination on substrates with bacteria. The results of the study concluded that the best explant length for making the artificial seeds was 5 mm; the best endosperm bead embedding plan was 3% sodium alginate+MSB （MS basic medium+organic components of B5 medium）+0.5 mg/L NAA+0.5 mg/L IBA， solidified in 100 mmol/L CaCl2 for 20 min; and the best formula of artificial seed coat was 4% sodium alginate+1% methylcellulose+1.0% sodium benzoate+0.02-0.04 mg/L penicillin+0.5 mg/L paclobutrazol， solidified in 200 mmol/L CaCl2 for 20 min. The artificial seeds had an aseptic germination rate of 95.0% and a rooting rate of 35.8%， and they could germinate into seedlings in an artificial substrate with bacteria. As a result， a new way for producing artificial seeds using the vegetative buds of upland cotton is obtained. The artificial seeds produced by this technology have a higher germination rate and can form seedlings in an environment with bacteria.

Vitis  spp.

Vitis  spp. is one of the most important economic fruit tree crops in the world， with high self-abortion possibility and heterozygosity， and many varieties have no seeds. Therefore， artificial seed technology may provide a convenient and feasible way for the reproduction and preservation of grapes. Das  [11]  published a report on successful plant regeneration using grape embedded somatic embryos. Cotyledon-stage embryos derived from leaf explants were individually embedded in 2% alginate colloid， and successfully transformed into test-tube plantlets in a medium containing halved B5 microelements， complete MS trace salts， 3% sucrose and 2.9 μmol/L gibberella， with an agar concentration of 0.7%. Before embedding， the embryos were transferred to B5 complete medium containing ABA （0.04 μmol/L） for 4-6 weeks. The storage time could be extended to 90 d without affecting the transformation potential.

Citrus reticulata  Blanco

Citrus reticulata  Blanco is the most widely cultivated fruit tree in tropical and subtropical regions in the world. Citrus juice contains high-quality citric acid， and is a good source of vitamin C and flavonoids. Antonietta  et al.   [12]  tried to produce  C. reticulata  artificial seeds by embedding somatic embryos with alginate， and found that the artificial seeds produced with embedded somatic embryos and artificial endosperm containing GA3 had a higher rate of plant transformation on agar medium compared with those free of embedding using artificial endosperm without adding growth regulators. The embedded somatic embryos containing GA3 could be used for the preservation of somatic embryos， and no significant effect would be produced on their  transformation rate after storage at 4 ℃ for 30 d. For plant transformation rate， agar medium was superior to other substrates in terms of culture medium alone. Clark  [13]  studied the effects of different storage conditions on the transformation rates of embedded and unembedded somatic embryos of Kinnow mandarin. The embedded and unembedded embryos were buried in liquid paraffin and stored at room temperature，  4 ℃  and liquid nitrogen. The results showed that the plantlet formation rate of embedded embryos after 60 d of storage at room temperature was 18.05%， while the plantlet formation rate of unembedded somatic embryos after 45 d of storage was only  13.88%;  the plantlet formation rate of embedded somatic embryos after storage at 4 ℃ for 120 d， was 11.11%， while the plantlet formation rate of unembedded somatic embryos stored at 4 ℃ for 90 d was only 5.55%; and after 7 months of storage in liquid nitrogen， the plantlet formation rates of embedded embryos and unembedded embryos were 58.33% and 51.38%， respectively. It shows that liquid nitrogen storage has little effect on plantlet formation rate， and liquid nitrogen can be adopted for a long time of storage. Antonietta  et al.   [14]  studied the effects of bactericide PPM and fungicide thiophanate-methyl on the storage of embedded somatic embryos at different stages， and found that the embedded somatic embryos could not be stored for more than 60 d at 4 ℃， and the addition of thiophanate-methyl to artificial endosperm can maintain the expected rooting rate and plantlet formation rate in test tubes and in the soil at a high level， which might be related to the fact that refrigeration for a certain period of time helps somatic embryos to mature better， and to the accumulation of storage protein and carbohydrates and other substances. However， the high-concentration bactericide PPM used alone or in combination with fungicides had adverse effects on germination， rooting and plantlet formation rates.

Fragaria ananassa  Duch

Fragaria ananassa  Duch is a perennial herb with strong  adaptability，  short growth cycle， early fruiting， quick efficiency， and wide cultivation. It is cultivated in China from Heilongjiang in the north， Guangdong in the south， Xinjiang in the west， and Jiangsu and Zhejiang in the east.  F. ananassa  is rich in nutrition， and has high economic benefits. It has both edible value and ornamental value， and its development prospects are very broad. The development of strawberry artificial seeds has been carried out earlier in China. In 1995， Li  et al.   [15]  used strawberry varieties  "Baojiao  Zaosheng"， "Fengxiang" and "Chunxiang" as test materials， and found that the medium combination of MS+6BA  2.0 mg/L +IAA 0.1 mg/L was most conducive to the formation of lateral buds. Inducing calli with MS+6BA 1.0 mg/L+IAA 0.1 mg/L+2， 4-D 1.0 mg/L and test tube plantlets and immature seeds as explants could give a higher induction rate. It can be used as the first step to induce embryoid bodies. For artificial endosperm formulas， 5 kinds of endosperm formulas were compared with adventitious buds as embedded bodies， and the combination of 1/2 MS+6BA 0.5 mg/L+IAA 0.5 mg/L had the highest germination rate， and can maintain nutrition supply for about 20 d  after  germination.

Ziziphus jujuba  Mill

Z. jujuba  Mill originated in China and is rich in vitamin C and vitamin P. In addition to serving as a fresh food， it can often be made into preserved fruit such as candied dates， red dates， smoked dates， black dates and wine dates， as well as raw materials for the food industry， such as jujube paste， jujube noodles， jujube wine and jujube vinegar. Hu  et al.   [16]  carried out the research and development of artificial seeds for  Z. jujuba  trees， which provides new research ideas for solving a series of urgent problems， including  Z. jujuba  branch cuttings that are extremely difficult to root， few new jujube shoots that can be used as scions on trees and low reproduction speed. They induced adventitious buds by callus culture of the jujube cultivar "Jidanzao"， and when the adventitious buds grew to 1-2 cm， the top 3-4 mm was cut  out as an embedded propagule. Sodium alginate （mass concentration  40 g/L） was dissolved in the endosperm medium composed of MS basic medium components and hormones， and the obtained mixture was sterilized at 121 ℃ for 20 min to prepare an aseptic embedding material. In an ultra-clean workbench， about 40 ml of the embedding material was transferred into a 50 ml beaker containing propagules， and one propagule was pipetted together with the embedding material using a pipette with an inner diameter of 4 mm， and dropped into the CaCl2 solution. The volume of each drop was 0.5 ml. After 5 min， the droplets were solidified into artificial seeds. The artificial seeds were taken out and rinsed with sterile water.

Carica papaya  L.

Papayas （ Carica papaya  L.） are also known as Shigua， Wanshouguo， Pengshengguo， Rugua. Papaya trees are small evergreen trees. Papayas are a tree species native to southern Mexico and adjacent central America. It is distributed in tropical and subtropical regions of the world. In China， it is mainly distributed in Guangdong， Hainan， Guangxi， Yunnan， Fujian， Taiwan and other provinces （regions）. Papayas have a wide range of uses， and are both as a fruit tree and as a raw material crop. Green papaya fruit is rich in papain and widely used in medicine， food， leather， textile and beauty， and serves as the main raw material of papain industry. Huang  et al.   [17]  induced the seedling radicle explants of hybrid papaya "Yuanyou No. 1" and obtained mature somatic embryos， which were embedded in sodium alginate， obtaining the artificial seeds which were germinated on hormone-free medium and formed plantlets， which were transplanted to the field and survived. It was found from the morphological observation of the plants that the artificial seed plants and the natural seed plants were not significantly different in shape and tree body， and the experiment initially proved that there was no variation in papaya.

Apple

In order to develop artificial apple seeds， Dakedong  et al.   [18]  used the young leaves of apple variety "Gala" as explants to culture somatic embryos， and on this basis， they explored the production process of artificial apple seeds. They divided the first three unfolded leaves from the top of the test-tube plantlets of apple variety "Gala" into three areas： upper， middle and lower from the leaf tip to the leaf base， and each area was divided into left and right sub-areas by leaf veins， forming 6 sub-areas in total. Each leaf was punctured 1 point at the center of 1 sub-area with a dissecting needle. Then， these leaves were inoculated on the medium of MS+BA 1.0 mg/L+NAA 4.0 mg/L+2， 4-D 0.5 mg/L+sucrose 20 g/L， and after culturing in the dark for 7 d， they were transferred to MS+BA 1.0 mg/L+sucrose 20 g/L medium to continue culture in the dark. After 40 d， 85% of the leaves developed direct somatic embryos around the wound. When making artificial seeds， the encapsulation matrix of somatic embryos was 4% sodium alginate （ added with MS+BA 0.4 mg/L+IAA 0.1 mg/L medium and 3% sucrose as artificial endosperm）， and the complexing electrolyte was 2%CaCl2， which was sterilized under high temperature and high pressure before use. Under sterile  conditions，  the somatic embryos were fully mixed with the wrapping medium in a small beaker and soaked for 3 to 4 min， and then， the inclusions was transferred into the CaCl2 solution one by one to react for 10 to 15 min using a glass rod. Finally， the CaCl2 solution was poured off， and the reaction was terminated by rinsing with sterile water 3 to 4 times， that is， the production of artificial apple seeds was completed.

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