CN107410024B - Avocado callus induction method and method for promoting bud differentiation of avocado callus - Google Patents
Avocado callus induction method and method for promoting bud differentiation of avocado callus Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention provides an induction method of avocado callus and a method for promoting the differentiation of avocado callus. The induction method of the avocado callus comprises the following steps: inoculating the avocado explant into an induction culture medium, and culturing for 4-30 days under the conditions of illumination intensity of 1200-1700 Lx, photoperiod of 8-14 h/day and temperature of 22-28 ℃ to obtain callus, wherein the induction culture medium is an MS basal culture medium which contains 20-40 g/L of sucrose, 5-12 g/L of agar, 0-3.0 mg/L of naphthylacetic acid, 0.1-3.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 0.1-3.0 mg/L of 6-benzylaminopurine and has pH of 5.8-6.5. According to the induction method of the avocado callus provided by the invention, the low-concentration 6-benzylamino adenine and 2, 4-dichlorophenoxyacetic acid hormone are combined, so that the induction rate of the cultured avocado callus is high; the invention researches the explant types of leaves, stem segments, bud segments and the like, finds that the induction rate of the stem segment explant is up to more than 90 percent, and lays a foundation for further successfully inducing complete plants in the future.
Description
Technical Field
The invention relates to the technical field of plant propagation, in particular to an induction method of avocado callus and a method for promoting bud differentiation of avocado callus.
Background
Avocado (Persea American Mill) belongs to evergreen woody fruit trees in the genus of avocado of Lauraceae, has rich nutritive value, and has certain medicinal and health-care effects. The traditional Chinese pear breeding mode has low breeding coefficient, and plant diseases and insect pests limit the planting and development of the Chinese pear; this problem can be solved to some extent by tissue culture techniques. The tissue culture technology has the advantages of good benefit, multiple propagation modes, short culture period and the like, and is researched and applied to fruits such as bananas, papayas, strawberries and the like.
In foreign countries, most scholars use immature zygotic embryos (sexual embryos formed by the development of a zygote formed by the fusion of male and female gametes of a plant) as explants; inducing somatic embryos and realizing the regeneration of avocado plants through a direct/indirect somatic embryogenesis way. Carlos L Lopez Encina et al, discussed the effects of different culture methods and different varieties on the induction of somatic embryo regeneration plants, using immature zygotic embryos of "Reed", "Hass", "Duke 7" and "A10" avocado varieties as explants. The results show that the somatic embryo incidence rate of 'Reed' avocado (Persea American Mill. cv. Reed) is high, and the two-step culture method (solid culture-liquid culture-solid culture) and the addition of 0.4g/L proline or 1g/L glutamine in the culture medium are beneficial to the maturation and germination of the somatic embryo; but the probability of bipolar growth of the somatic embryo is still low (shoot and root growth at the same time). Rohim discusses the influence of embryo age, culture medium type and sucrose concentration on the maturation and germination of somatic embryos, and finds that the best effect is achieved when the embryo age is 180d, MS is a minimal medium and the sucrose concentration is 30 g/L. But the related research of inducing the whole plant of the avocado by taking the leaves, the stem sections and the like as explants through a direct or indirect organogenesis way is less. At present, only the Young takes leaves and stems of the Lula variety of the avocado as explants to study the influence of single hormone species, temperature and illumination on callus induction; however, callus bud differentiation was not further investigated. Barrera-Guerra et al induced the whole plant of avocado by direct organogenesis using the stem of avocado as explant, and investigated the influence of growth hormone species and its concentration on rooting culture. The indirect rooting method is found to have good effect, but the rooting rate is still low.
In China, the research on the tissue culture of the avocado is less, and only people with the heights of longeyana, pengzhi jade tablet, and He bizu have carried out related research on the avocado at present. Liangri Gao and He Bizhu et al use the stem segment of adult avocado tree as explant to induce cluster buds via direct organogenesis and further realize avocado plant regeneration. The hypocotyl of pengming jade tablet is used as the explant induced callus and the low temperature freezing preservation technology is combined to discuss the relevant factors influencing the germ plasm preservation.
At present, the influence of different hormone types, concentrations and explant types on the induction and bud differentiation of the calluses of the Haas avocado is not reported to be researched so as to screen out the optimal induction conditions and explants.
Disclosure of Invention
In order to overcome the defects, the invention provides an induction method of avocado callus and a method for promoting bud differentiation of avocado callus.
The technical scheme adopted by the invention for solving the technical problem is as follows:
in a first aspect, the invention provides a method for inducing avocado callus, which comprises the following steps:
inoculating the avocado explant into an induction culture medium, and culturing for 4-30 days under the conditions of illumination intensity of 1200-1700 Lx, photoperiod of 8-14 h/day and temperature of 22-28 ℃ to obtain callus, wherein the induction culture medium is an MS basal culture medium which contains 20-40 g/L of sucrose, 5-12 g/L of agar, 0-3.0 mg/L of naphthylacetic acid, 0.1-3.0 mg/L of 2, 4-dichlorophenoxyacetic acid, 0.1-3.0 mg/L of 6-benzylaminopurine and has pH of 5.8-6.5.
Preferably, the avocado explant comprises one or more of avocado leaves, avocado petioles, avocado stem tips, avocado stem segments and avocado bud segments.
Further preferably, the avocado explant is an avocado stem segment.
Preferably, the variety of the avocado includes, but is not limited to, one of "Haas" avocado, "Reed" avocado, "Duke 7" avocado, "A10" avocado.
Further preferably, the variety of the avocados is 'hass' avocados.
Preferably, the cultivation time of the avocado callus is 6-14 days.
Preferably, the induction culture medium is an MS basal culture medium which contains 20-40 g/L of sucrose, 5-12 g/L of agar, 0.1-3.0 mg/L of 2, 4-dichlorophenoxyacetic acid and 0.1-3.0 mg/L of 6-benzylamino adenine and has the pH value of 5.8-6.5.
More preferably, the induction culture medium is an MS basal culture medium which contains 20-40 g/L of sucrose, 5-12 g/L of agar, 0.1-2.0 mg/L of 6-benzylamino adenine and 0.1mg/L of 2, 4-dichlorophenoxyacetic acid and has the pH value of 5.8-6.5.
More preferably, the induction medium is an MS basal medium which contains 20-40 g/L of sucrose, 5-12 g/L of agar, 0.1-2.0 mg/L of 6-benzylamino adenine and 0.1mg/L of 2, 4-dichlorophenoxyacetic acid and has the pH value of 5.8-6.5.
More preferably, the induction culture medium is an MS basal culture medium which contains 30g/L of sucrose, 7g/L of agar, 0.5mg/L of 6-benzylamino adenine and 1.0mg/L of 2, 4-dichlorophenoxyacetic acid and has the pH value of 5.8-6.5.
Preferably, the explant is prepared by sterilizing the collected avocado sample.
Further preferably, the explant is prepared by the following method:
putting the collected avocado sample on a super-clean workbench, adding alcohol to sterilize the surface for about 15-25 s, cleaning with sterile water for 2-3 times, and transferring into HgCl2Sterilizing the solution for about 4-5 min, and washing with sterile water for 4-5 times; placing the treated stem segments of the avocados on sterile filter paper to absorb water, and preparing explants; wherein the concentration of the alcohol is not lower than 70%; the HgCl2The concentration of the solution is 0.05-1%.
Preferably, in the method for inducing the avocado callus, the inductivity of the avocado callus is not less than 75%.
In a second aspect, the invention also provides a method for promoting the differentiation of avocado callus buds, which comprises the following steps:
taking the callus cultured by the avocado callus induction method in the first aspect, inoculating the obtained callus into a bud differentiation culture medium and culturing for 30-50 days, wherein the bud differentiation culture medium is an MS basal medium which contains 20-40 g/L of sucrose, 5-12 g/L of agar, 0.1-3.0 mg/L of naphthylacetic acid and 0.1-3.0 mg/L of 6-benzylamino adenine and has the pH value of 5.8-6.5.
Preferably, in the method for promoting the bud differentiation of the avocado callus, the germination rate of the avocado callus is not less than 35-60%.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the following beneficial effects:
in the prior art, multiple hormone components are often adopted to induce callus, or high-concentration naphthylacetic acid, 2, 4-dichlorophenoxyacetic acid or the combination of two hormones are adopted to induce callus, but the induction method of avocado callus provided by the invention adopts low-concentration 6-benzylaminopurine and 2, 4-dichlorophenoxyacetic acid hormone components to combine, so that the induction rate of the cultured avocado callus is high; the invention researches the types of explants such as leaves, stem segments, bud segments and the like, finds that the induction rate of the stem segment explant is up to more than 90 percent, the bud ratio is high, and lays a foundation for further successfully inducing complete plants in the future.
Drawings
FIG. 1 shows the dry and fresh weight of callus induced by leaves of "Haas" avocado at different concentrations of NAA provided in the examples of the present invention;
FIG. 2 shows the dry and fresh weight of callus induced by leaves of "Haas" avocado at various concentrations of 2,4-D according to the present invention;
FIG. 3 shows the dry and fresh weight of callus induced by leaves of "Haas" avocado at different concentrations of 6-BA according to the present invention;
FIG. 4 shows the dry and fresh weight of callus induced by explants at different parts of "Haas" avocado provided by the present invention;
FIG. 5 shows the growth of calluses of "Haas" avocado according to the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments.
It is understood that Excell software is used to collate and calculate the data, the average value represents the overall level of each treatment, and ANOVA of SPSS19.0 statistical software is used to analyze the difference of each parameter. The callus induction rate, water content and water content are calculated according to the following formula:
callus induction rate (number of explants forming callus/total number of explants inoculated) 100%;
water content (g) fresh-dry weight;
the moisture content of the callus is (water content of the callus/fresh weight of the callus) 100%;
the germination rate (number of germinated callus pieces/inoculated callus pieces) is 100%.
Example 1 Effect of different Single hormone classes and concentrations thereof on the Induction of callus from leaves of "Haas" avocado
The embodiment of the invention provides a method for inducing a Huas avocado callus, which comprises the following steps:
(1) and (3) explant disinfection and sterilization: putting the collected Haas avocado leaves on a super clean workbench, adding 75% alcohol for surface disinfection for about 15-25 s, cleaning with sterile water for 2-3 times, and transferring to 0.1% HgCl2Sterilizing the solution for about 4-5 min, and washing with sterile water for 4-5 times; placing the explant on sterile filter paper to absorb water, cutting off the tail part and the edge part of the leaf under the condition of sterile environment, and leaving the part close to the main vein to be cut into 5 x 5 mm; preparing an explant for later use;
(2) callus culture: respectively inoculating the explants obtained in the step (1) into induction culture media added with (0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0mg/L) naphthylacetic acid (NAA) and (0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0mg/L)2, 4-dichlorophenoxyacetic acid (2,4-D) with different concentrations, wherein the induction culture media are MS culture media basis, further comprise 30g/L sucrose and 7g/L agar, and the pH is 6.0 +/-0.1. And inoculating about 30 explants per treatment, repeating for 3 times, and inoculating 2-3 explants per bottle. Callus was obtained after 30 days of cultivation. Wherein the culture conditions are as follows: culturing in tissue culture room with illumination intensity of 1700Lx, photoperiod of 14 h/day (10 h dark each day) and temperature of 25 + -2 deg.C; regularly observing, counting the callus induction rate and dry fresh weight, and screening the optimal callus induction culture medium.
And (3) analyzing an experimental result:
(1) the results were observed periodically: inoculating leaf explants into induction culture media containing NAA and 2,4-D with different concentrations, inoculating for about 2 weeks, and allowing different degrees of enlargement at the incision to form callus visible to naked eyes. The main distribution is at two ends of the cut of the explant, and a few are scattered on the surface of the explant, so the explant is small in volume, milky white, hard in texture and mostly in the shape of a particle ball (see fig. 5A and B). If the culture time is too long, the formed callus is easy to brown and die.
(2) The effect of different hormone classes and their concentrations on callus induction of "hass" avocado leaves is seen in table 1 and figures 1 and 2.
As can be seen from Table 1, the effect of different hormones and their concentrations on callus induction was significant. In the medium without any hormones, no change of the explants occurred. In the culture medium containing NAA and 2,4-D with different concentrations, part of the culture medium can induce callus. The induction effect is better when 2,4-D is added, the induction rate is 10.6-45.3%, and the highest induction rate is 45.3% when the concentration of the 2,4-D is 1.0 mg/L. The callus induction rate increased and then decreased with the increase of the 2,4-D concentration, indicating that the high concentration of 2,4-D is not favorable for the induction of callus. When NAA is added into the culture medium, the inductivity is 5.7-20.2%; when the concentration of NAA is 1.0mg/L, the induction rate is up to 20.2%. Along with the increase of the concentration of NAA, the induction rate is increased and then decreased, which indicates that the high concentration of NAA is not beneficial to the induction of the callus.
It is seen from FIGS. 1 and 2 that with increasing hormone concentration, the fresh and dry weight of the callus formed increased and then decreased. The fresh weight reached a maximum at a 2,4-D and NAA concentration of 1.0mg/L, which was 0.83g and 0.37g, respectively.
In general, when a single growth hormone is added, the induction effect of the callus of the leaf of the 'hass' avocado is not ideal, and the effect of the callus can be improved when the callus is possibly used in combination with cytokinin, which needs to be further researched and discussed.
TABLE 1 Effect of different hormone classes and concentrations on callus induction of "Haas" avocado leaves
EXAMPLE 2 Effect of different hormone combinations on the Induction of callus from leaves of "Haas" avocado
To further illustrate the advantageous effects of the present invention, the procedure of example 1 of the present invention was repeated to replace the hormone components in the induction medium in step (2) of example 1 of the present invention with the hormone components shown in Table 2.
And (3) analyzing an experimental result:
(1) the results were observed periodically: the leaves are respectively inoculated into 6 culture mediums, and after about 6 days of inoculation, the explants begin to expand and dedifferentiate at the incision parts to form pale yellow callus. Along with the prolonging of the culture time, the formed callus gradually expands and slowly covers the explant; callus was formed at the latest around inoculation 14 d. The formed callus is loose, yellowish white and has more water content; part of the callus formed was accompanied by flocculent white matter (see FIG. C), and part of the explants without callus formed enlarged at the incision and appeared browned. Counting and transferring are started after inoculation for about 30d, and the callus is easy to brown when the culture time is too long.
(2) As shown in Table 2, the callus can be induced by adding 0.1-2.0 mg/L6-BA, 1.0 mg/L2, 4-D and 0.1mg/L LNAA into the culture medium, and the effect is the best when the concentration of 6-BA is 0.5mg/L, and the induction rate is 55.8%; the induction effect in the MS +0.5 mg/L6-BA +1.0 mg/L2, 4-D +0.1mg/LNAA culture medium is equivalent to that of the MS +0.5 mg/L6-BA +1.0 mg/L2, 4-D culture medium; however, the callus induction effect in MS +0.1 mg/L6-BA +1.0 mg/L2, 4-D culture medium is poor, and the browning of the callus formed by induction is serious.
As can be seen from FIG. 3, the fresh weight of the formed callus increased first and then decreased with the increase of the concentration of 6-BA, indicating that the high concentration of 6-BA is not favorable for the induction of callus. The water content of the callus is 93-95.9%, the fresh weight of the callus is 3.82g when the concentration of 6-BA is 0.5mg/L, the fresh weight of the callus formed by induction in an MS +0.5 mg/L6-BA +1.0 mg/L2, 4-D +0.1mg/LNAA culture medium is 0.315g, and the difference between the two is small; it is demonstrated that the addition of 0.1mg/L NAA to MS +0.5 mg/L6-BA +1.0 mg/L2, 4-D medium is not very effective.
TABLE 2 Effect of different media on callus induction of "Haas" avocado leaves
Example 3 Effect of different explant types on callus induction of "Haas" avocado
To further illustrate the beneficial effects of the present invention, the procedure of example 1 of the present invention was repeated, and "avocado leaves" in step (1) of example 1 of the present invention was replaced with explants as shown in Table 3.
(1) The results were observed periodically: the leaf, petiole, stem tip and stem segment (with/without effective bud) of avocado are inoculated into MS +0.5 mg/L6-BA +1.0 mg/L2, 4-D minimal medium, and the induction time, texture, color and the like of callus are observed. The explants begin to expand after about 4 days of inoculation, and visible callus can be seen after about 7 days; the formed callus is milky white, loose and has high water content (see FIGS. 5D-K), and the callus formed by partial explants is accompanied by white floccules (see FIG. 5F, G, H, K)
(2) As shown in Table 3, the leaf, petiole, stem tip and stem segment (with/without effective bud) all induced callus, and the influence of the explant type on callus induction was significant. The induction effect of the stem segment is optimal, the induction rate of the stem segment without the effective bud point is 98.3 percent, and the induction rate of the stem segment with the effective bud point is 72.7 percent; it is likely that the callus formed from the stem segments with efficient shoot points is distributed sporadically on the surface of the explant, and is accompanied by elongation and growth of axillary buds in addition to callus formation, thereby consuming part of the nutrients. The worst induction effect of 5 explant types is the stem tip, the induction rate is 20%, and the culture medium and culture conditions are possibly unfavorable for inducing callus.
FIG. 4 shows that the average fresh weight of the callus induced by leaves, petioles, stem tips and stem segments (with/without effective bud points) is 2.25-3.415 g, the average dry weight is 0.13-0.15 g, and the water content is 93.9-96.2%. The fresh weight of the explant was the greatest when it was a stem without a viable shoot point, 3.415 g.
TABLE 4 Effect of different part explants on callus induction of "Haas" avocado
EXAMPLE 4 Effect of different callus culture times on sprouting Rate
The embodiment of the invention provides a method for improving induction and growth of avocado callus, which comprises the following steps:
(1) and (3) explant disinfection and sterilization: putting the collected stem segments (without effective bud points) of the Haas avocado on a super clean workbench, adding 75% alcohol for surface disinfection for about 15-25 s, cleaning with sterile water for 2-3 times, and transferring to 0.1% HgCl2Sterilizing the solution for about 4-5 min, and washing with sterile water for 4-5 times; placing the treated stem segments of the avocados on sterile filter paper to absorb water, and preparing explants for later use;
(2) callus culture: respectively inoculating the explants obtained in the step (1) into an induction culture medium, wherein the induction culture medium is an MS basal culture medium which contains 30g/L of sucrose, 7g/L of agar, 0.5mg/L of naphthylacetic acid (NAA) and 1.0mg/L of 2, 4-dichlorophenoxyacetic acid (2,4-D) and has the pH value of 6.0 +/-0.1; and inoculating about 30 explants per treatment, repeating for 3 times, and inoculating 2-3 explants per bottle. Callus was obtained after 30 days of cultivation. Wherein the culture conditions are as follows: culturing in a tissue culture room with illumination intensity of 1500Lx, illumination for 12 h/darkness for 12h and temperature of 25 + -2 deg.C in the dark; regularly observing, counting the induction rate and dry fresh weight of the callus, and respectively culturing for 5, 7, 10, 14, 18, 21 and 30 days to obtain the callus;
(3) bud differentiation culture: respectively inoculating the calluses obtained in the step (2) into bud differentiation culture media, wherein the bud differentiation culture media are MS basal culture media containing 30g/L of sucrose, 7g/L of agar, 0.5mg/L of naphthylacetic acid (NAA) and 1.0mg/L of 6-benzylamino adenine (6-BA) and the pH value of the MS basal culture media is 6.0 +/-0.1; 30 explants were inoculated per treatment, repeated 3 times, observed for growth and counted around 40 days.
And (3) analyzing an experimental result: as shown in Table 5, the callus obtained after 30 days of cultivation was browned and died to a different extent by bud differentiation cultivation and was hard to differentiate and bud, while the callus obtained after 6, 10, 14, 18 days of cultivation was not browned or has a small degree of browning, and the bud ratio was much higher than that of the callus obtained after 21 and 30 days of cultivation.
TABLE 5 Effect of different callus culture times on germination Rate
EXAMPLE 5 Effect of the same Induction Medium on callus induction of explants of different varieties
(1) And (3) explant disinfection and sterilization: respectively collecting the stem sections (without effective bud points) of the following varieties of fruit trees: putting ' Haas ' avocado, Lula ' avocado, ' Duke 7 ' avocado and ' Reed ' avocado on a clean bench, adding 75% alcohol to sterilize the surface for about 15-25 s, cleaning with sterile water for 2-3 times, and transferring into 0.1% HgCl2Sterilizing the solution for about 4-5 min, and washing with sterile water for 4-5 times; placing the explant on sterile filter paper to absorb water, cutting off the tail part and the edge part of the leaf under the condition of sterile environment, and leaving the part close to the main vein to be cut into 5 x 5 mm; preparing a Hass avocado leaf explant, an avocado leaf explant, a Lula avocado leaf explant, a Duke 7 avocado leaf explant and a Reed avocado leaf explant for later use;
(2) callus culture: respectively inoculating the explants obtained in the step (1) into an induction culture medium, wherein the induction culture medium is an MS basal culture medium which contains 30g/L of sucrose, 7g/L of agar, 0.5mg/L of naphthylacetic acid (NAA) and 1.0mg/L of 2, 4-dichlorophenoxyacetic acid (2,4-D) and has the pH value of 6.0 +/-0.1. About 30 explants were inoculated per treatment, which was repeated 3 times, 3 explants per flask. Callus was obtained after 30 days of cultivation. Wherein the culture conditions are as follows: culturing in a tissue culture room with illumination intensity of 1700Lx, illumination for 14 h/dark for 10h and temperature of 25 +/-2 ℃ in the dark; and (5) regularly observing and counting the callus induction rate.
And (3) analyzing an experimental result: as shown in Table 5, the different varieties of explants have obvious induction rate difference of callus under the same cultivation condition, and the induction rate of the explant of the stem segment of the "Haas" avocado of the embodiment is the highest, while the induction rate of the explants of other varieties is far lower than that of the "Haas" avocado, which fully indicates that the different genotypes of explants have different sensitivity to plant growth regulators, and the different genotypes of explants have different capabilities of inducing callus and are possibly related to the inherent hormone content.
TABLE 6 influence of the same Induction Medium on callus induction of explants of different varieties
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (4)
1. The method for inducing the avocado callus is characterized by comprising the following steps of:
inoculating an avocado explant into an induction culture medium, and culturing for 6-14 days under the conditions of illumination intensity of 1200-1700 Lx, photoperiod of 8-14 h/day and temperature of 22-28 ℃ to obtain callus, wherein the induction culture medium is an MS basal culture medium which contains 30g/L of sucrose, 7g/L of agar, 1.0mg/L of 2, 4-dichlorophenoxyacetic acid and 0.5mg/L of 6-benzylaminopurine and has pH of 6.0; the avocado explant is an avocado stem section without a bud point; the variety of the avocados is 'Haas' avocados; the inductivity of the avocado callus is more than 90%.
2. The method for inducing avocado callus according to claim 1, wherein said explant is obtained by sterilizing a harvested avocado sample.
3. A method for promoting the differentiation of avocado callus buds is characterized by comprising the following steps:
taking the callus cultured by the method for inducing avocado callus according to any one of claims 1 to 2, and inoculating the callus obtained into a bud differentiation medium comprising an MS basal medium containing 30g/L sucrose, 7g/L agar, 0.5mg/L naphthylacetic acid, 1.0 mg/L6-benzylaminoadenine and having a pH of 6.0, and culturing for 30 to 50 days.
4. The method for promoting the bud differentiation of avocado callus according to claim 3, wherein the germination rate of avocado callus is not less than 35-60%.
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| Avocado Callus And Bud Culture;M.J.Young;《Proc.Fla.State Hort.Soc》;19831231;第96卷;第1页材料与方法至表8 * |
| M.J.Young.Avocado Callus And Bud Culture.《Proc.Fla.State Hort.Soc》.1983,第96卷第181-182页. * |
| 油梨组培快繁技术研究;梁日高等;《中国南方果树》;20081231;第37卷(第3期);第52页第1节，第2.2节 * |
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